Protein antigens that provide protection against pneumococcal colonization and/or disease

ABSTRACT

The present application is generally directed to novel pneumococcal polypeptide antigens and nucleic acids encoding such antigens, and immunogenic compositions comprising such antigens for treating and preventing pneumococcal infection. The present invention further provides method of using the antigens to elicits an immune response (e.g., IL-17A response, a T cell-mediated and/or B-cell-mediated immune responses). The present invention also provides methods of prophylaxis and/or treatment of pneumococcal-mediated diseases, such as sepsis, comprising administering an immunogenic composition including one or more of a combination of pneumococcal antigens or functional fragments thereof as disclosed herein. In some embodiments, one or more pneumococcal antigens can be present in a polysaccharide conjugate. The compositions induce an anti-pneumococcus immune response when administered to a mammal. The compositions can be used prophylactically to vaccinate an individual and/or therapeutically to induce a therapeutic immune response to an infected individual.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 371 National Phase Entry of International PatentApplication No. PCT/US2014/015254 filed on Feb. 7, 2014 which claimsbenefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent ApplicationSer. No. 61/762,062 filed Feb. 7, 2013, the contents of which areincorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 6, 2014, isnamed 701039-074841-PCT_SL.txt and is 640,188 bytes in size.

FIELD OF THE INVENTION

The present application is generally directed to methods for identifyingimmunogens from organisms and pathogens, and in particular foridentifying immunogens which when administered as vaccines elicit acellular and/or humoral immune response. The present application is alsodirected to pneumococcal T-cell immunogens, and methods and compositionsthereof.

BACKGROUND OF THE INVENTION

Almost one million children in the developing world die of pneumococcalinfections each year. Despite the effectiveness of the conjugatepneumococcal vaccines, problems with this approach remain including theexpense of production and delivery, and resulting serotype replacementas demonstrated in several clinical trials and epidemiologic studies.One positive effect of the current capsular-based vaccines has beenevident in the patient population that is not being vaccinated: herdimmunity plays an impressive role in the current vaccine strategy. Foreach case of pneumococcal disease prevented in children, about threecases of pneumococcal disease are prevented in adults by herd immunity.In this context at least, prevention of pneumococcal colonization is amain goal of protein-based vaccine approaches, because blockingcolonization will block disease.

Alternative pneumococcal vaccines that elicit serotype-independentimmunity, and that maybe more readily available to economically emergingcountries are needed urgently. New antigens that can address this needwould be very attractive. Additionally, current methods to identifyimmunogens focus on techniques that do not fully optimize the extractionor identification of the full antigen repertoire. This is true in thecase of pneumococcus as well as other pathogens. A method that canidentify a new set of antigens has potential to be impactful for thedevelopment of vaccines for a wide set of pathogens, includingpneumococcus.

SUMMARY OF THE INVENTION

An aspect of the present invention encompasses the discovery of novelantigens for pneumococcus that elicit antigen specific immune responsesin mammals. Such novel antigens, and/or nucleic acids encoding theantigens, can be incorporated into immunogenic compositions andadministered to elicit immune responses, e.g., to provide protectionagainst pneumococcal colonization, invasive diseases, such as but notlimited to, sepsis, and diseases and disorders caused by pneumococcus.Such novel antigens and/or responses to novel antigens can be detectedto identify and/or characterize immune responses to pneumococcus.

In one aspect of the present invention provides immunogenic compositions(e.g., vaccines) comprising at least one isolated pneumococcal antigenselected from the pneumococcal proteins SP0010, SP0043, SP0079, SP0084,SP0092, SP0098, SP0106, SP0107, SP0127, SP0149, SP0191, SP0198, SP0249,SP0321, SP0346, SP0402, SP0453, SP0564, SP0582, SP0589, SP0601, SP0604,SP0617, SP0620, SP0629, SP0648, SP0659, SP0662, SP0664, SP0678, SP0724,SP0742, SP0757, SP0785, SP0787, SP0872, SP0878, SP0899, SP1002, SP1026,SP1032, SP1069, SP1154, SP1267, SP1376, SP1386, SP1404, SP1405, SP1419,SP1479, SP1500, SP1545, SP1560, SP1624, SP1652, SP1683, SP1826, SP1872,SP1891, SP1897, SP1942, SP1966, SP1967, SP1998, SP2048, SP2050, SP2083,SP2084, SP2088, SP2145, SP2151, SP2187, SP2192, SP2197, SP2207 andSP2218, or fragments thereof.

One aspect of the present invention relates to an immunogeniccomposition comprising at least one isolated pneumococcal antigen orfragment thereof with the amino acid sequence selected from SEQ ID NO:1-76 or SEQ ID NO: 153-234, and wherein the composition elicits animmune response against Streptococcus pneumoniae when administered to amammal. In some embodiments, a pneumococcal antigen or fragment thereofexists as a fusion conjugate, for example, where the fusion conjugate isa polysaccharide conjugate. In some embodiments, a fusion conjugatecomprises the pneumococcal antigen or fragment thereof fused to apneumococcal pneumolysoid PdT, wherein the pneumococcal pneumolysoid PdTis conjugated to the polysaccharide. In some embodiments, the fusionconjugate comprises a polysaccharide which is dextran, Vi polysaccharideof Salmonella typhi, or pneumococcal cell wall polysaccharide (CWPS), oranother polysaccharide of prokaryotic or eukaryotic origin.

In some embodiments of all aspects of the invention, an immunogeniccomposition induces a IL-17A (Th17-cell) response in a subject. In someembodiments, an immunogenic composition induces an immune response whichcomprises a humoral immune response and/or cellular immune response.

In some embodiments, an immunogenic composition is further prepared as avaccine that reduces or protects a mammal against pneumococcalcolonization. In some embodiments, an immunogenic composition furthercomprises an adjuvant. In some embodiments, an immunogenic compositionas disclosed herein is administered mucosally.

In some embodiments of all aspects of the invention, an immunogeniccomposition comprises at least 3, or at least 5, or between 5-20pneumococcal antigens or fragments or more than 20 with the amino acidsequence selected from SEQ ID NO: 1-76 or SEQ ID NO: 153-234.

In some embodiments of all aspects of the invention, an immunogeniccomposition comprises at least one of the pneumococcal proteins SP0785,SP1500 and SP2145.

Another aspect of the present invention relates to a method of inducingan IL-17A response in a subject, comprising administering to the subjectat least one immunogenic composition as disclosed herein, e.g., apneumococcus antigen of Table 1, or a functional fragment thereofeffective to induce an immune response against Streptococcus pneumoniaein the subject.

Another aspect of the present invention relates to a method to protectagainst pneumococcus colonization, comprising administering to thesubject at least one immunogenic composition as disclosed herein, e.g.,a pneumococcal antigen of Table 1, or a functional fragment thereof.

Another aspect of the present invention relates to a method to elicit animmune response against Streptococcus pneumoniae in a mammal, the methodcomprising administering to the mammal at least one immunogeniccomposition comprising one or more isolated pneumococcal antigen orfragment thereof as disclosed herein in Table 1, with the amino acidsequence selected from SEQ ID NO: 1-76 or SEQ ID NO: 153-234 in aneffective to induce an immune response against Streptococcus pneumoniaein the subject.

Another aspect of the present invention relates to a method to protectagainst Salmonella typhi colonization in a mammal, comprisingadministering to the mammal at least one immunogenic composition asdisclosed herein, e.g., a pneumococcus antigen of Table 1, or afunctional fragment thereof effective to induce an immune responseagainst Streptococcus pneumoniae in the subject.

In all embodiments of the aspects described herein, an immunogeniccomposition can comprise at least one isolated pneumococcus antigen orfragment thereof selected from SP0785 or SP1500, and/or has an aminoacid sequence substantially identical to SEQ ID NO: 34 (SP0785) or SEQID NO: 51 (SP1500) or a functional fragment thereof.

Another aspect of the present invention relates to a method to protectagainst an invasive disease of Streptococcus pneumoniae in a subject,comprising administering to the subject an immunogenic composition asdisclosed herein, e.g., a pneumococcus antigen of Table 1, or afunctional fragment thereof in effective to induce an immune responseagainst Streptococcus pneumoniae in the subject. In some embodiments,such an invasive disease is, for example, sepsis. In such an embodiment,the method comprises an immunogenic composition comprising at least oneisolated pneumococcus antigen or fragment thereof is SP1386, SP1500,SP0084 and SP1479 and SP0346, and/or at least one pneumococcal antigenthat has an amino acid sequence substantially identical to SEQ ID NO: 46(SP1386), SEQ ID NO: 51 (SP1500), SEQ ID NO: 4 (SP0084), SEQ ID NO: 50(SP1479) and SEQ ID NO: 15 (SP0346) or a functional fragment thereof.

In all embodiments of the aspects described herein, an immunogeniccomposition can be administered by mucosal administration, or any otherapplicable route, for example but not limited to, intravenous,subcutaneous or intraperitoneal (IP) administration.

In some embodiments, an immunogenic composition comprises at least oneisolated pneumococcus antigen selected from any one or a combination ofthe pneumococcus antigens with the amino acid sequences 1-76 or SEQ IDNO: 153-234. In some embodiments, the pneumococcus antigens are the fulllength pneumococcus proteins of SP0010, SP0043, SP0079, SP0084, SP0092,SP0098, SP0106, SP0107, SP0127, SP0149, SP0191, SP0198, SP0249, SP0321,SP0346, SP0402, SP0453, SP0564, SP0582, SP0589, SP0601, SP0604, SP0617,SP0620, SP0629, SP0648, SP0659, SP0662, SP0664, SP0678, SP0724, SP0742,SP0757, SP0785, SP0787, SP0872, SP0878, SP0899, SP1002, SP1026, SP1032,SP1069, SP1154, SP1267, SP1376, SP1386, SP1404, SP1405, SP1419, SP1479,SP1500, SP1545, SP1560, SP1624, SP1652, SP1683, SP1826, SP1872, SP1891,SP1897, SP1942, SP1966, SP1967, SP1998, SP2048, SP2050, SP2083, SP2084,SP2088, SP2145, SP2151, SP2187, SP2192, SP2197, SP2207 and SP2218. Insome embodiments, a pneumococcus antigen comprises a pneumococcusprotein that lacks a signal sequence and/or transmembrane domain. Insome embodiments, a pneumococcus antigen comprises a mixture of a fulllength pneumococcus proteins and fragments resulting from processing, orpartial processing of a signal sequence by an expression host, e.g., E.coli or an insect cell line (e.g., the baculovirus expression system),or a mammalian (e.g., human or Chinese hamster Ovary (CHO)) cell line.As used herein, the terms “portion” and “fragment” or grammaticalequivalents are used interchangeably.

In some embodiments, the pneumococcal antigens are fragments of the fulllength pneumococcal proteins of SP0010, SP0043, SP0079, SP0084, SP0092,SP0098, SP0106, SP0107, SP0127, SP0149, SP0191, SP0198, SP0249, SP0321,SP0346, SP0402, SP0453, SP0564, SP0582, SP0589, SP0601, SP0604, SP0617,SP0620, SP0629, SP0648, SP0659, SP0662, SP0664, SP0678, SP0724, SP0742,SP0757, SP0785, SP0787, SP0872, SP0878, SP0899, SP1002, SP1026, SP1032,SP1069, SP1154, SP1267, SP1376, SP1386, SP1404, SP1405, SP1419, SP1479,SP1500, SP1545, SP1560, SP1624, SP1652, SP1683, SP1826, SP1872, SP1891,SP1897, SP1942, SP1966, SP1967, SP1998, SP2048, SP2050, SP2083, SP2084,SP2088, SP2145, SP2151, SP2187, SP2192, SP2197, SP2207 and SP2218, forexample, at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 150, 200,250, 300, 350 or 400 consecutive amino acids of such proteins.

In some embodiments, a pneumococcal antigen comprises an amino acidsequence which is at least 60% (e.g., at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98% or at least 99%) identical to at 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 100, 150, 200, 250, 300, 350 or 400 consecutiveamino acids of the pneumococcal proteins of SP0010, SP0043, SP0079,SP0084, SP0092, SP0098, SP0106, SP0107, SP0127, SP0149, SP0191, SP0198,SP0249, SP0321, SP0346, SP0402, SP0453, SP0564, SP0582, SP0589, SP0601,SP0604, SP0617, SP0620, SP0629, SP0648, SP0659, SP0662, SP0664, SP0678,SP0724, SP0742, SP0757, SP0785, SP0787, SP0872, SP0878, SP0899, SP1002,SP1026, SP1032, SP1069, SP1154, SP1267, SP1376, SP1386, SP1404, SP1405,SP1419, SP1479, SP1500, SP1545, SP1560, SP1624, SP1652, SP1683, SP1826,SP1872, SP1891, SP1897, SP1942, SP1966, SP1967, SP1998, SP2048, SP2050,SP2083, SP2084, SP2088, SP2145, SP2151, SP2187, SP2192, SP2197, SP2207and SP2218.

Accordingly, the inventors herein have identified pneumococcal T-cellimmunogens that both induce an IL-17A (e.g. a Th17-cell) response andprotect mice from colonization. These proteins, including SP0785,SP1500, SP2145, or fragments thereof, show promise as vaccine candidatesagainst colonization and sepsis. In some embodiments, a novelpneumococcal immunogens as disclosed herein e.g., as disclosed in Table1, are administered by mucosal immunization, and can be optionallyadministered with an adjuvant, to reduce subsequent pneumococcal nasalcolonization.

In some embodiments, an immunogenic composition comprises a SP1386,SP1500, SP0084, SP1479 and SP0346, or fragments thereof in a method toprevent or treat sepsis in a subject, such as a mammalian subject,including a human subject.

In some embodiments, an immunogenic composition comprising at least oneisolated pneumococcal antigen is prepared as a vaccine that reduces orprotects a mammal against pneumococcal colonization. In someembodiments, such an immunogenic composition can further comprise atleast one adjuvant, e.g., selected from the group comprising, but notlimited to cholera toxin, CFA, IFA, alum and others commonly known inthe art and disclosed herein.

Another aspect of the present invention relates to pharmaceuticalcomposition for eliciting an immune response in a mammal comprising theT-cell stimulating immunogens prepared according to the methods asdisclosed herein. In such embodiments, a pharmaceutical composition canfurther comprise an adjuvant and/or a vaccine scaffold. In someembodiments, a pharmaceutical composition can further comprise anadjuvant.

In some embodiments, the immunogenic composition as disclosed herein canbe administered to a subject mucosally. In all aspects of allembodiments as described herein, a subject is a mammalian subject, e.g.,a human, however other subjects are contemplated such as domestic andagricultural animals and the like.

The invention also provides compositions including nucleic acidsencoding a pneumococcal antigen as described herein. In someembodiments, a composition includes an isolated nucleic acid comprisinga nucleotide sequence encoding a pneumococcal antigen selected fromTable 1, or combinations thereof, and further comprises apharmaceutically acceptable excipient. In some embodiments, acomposition further comprises an adjuvant.

In another aspect, the invention provides methods for eliciting animmune response against pneumococcus in a mammal based on nucleic acidsdescribed herein in Table 1. In some embodiments, the invention providesmethods for eliciting an immune response against pneumococcus in amammal by administering to the mammal a composition comprising a nucleicacid, wherein the nucleic acid comprises a nucleotide sequence encodinga pneumococcal antigen as disclosed in Table 1 or combinations thereof.

In another aspect, the invention provides methods for characterizingand/or detecting an immune response to a pneumococcal antigen in asubject (e.g., a pneumococcal polypeptide antigen selected from Table 1or combinations thereof). In some embodiments, an immune response in anaive subject is characterized. In some embodiments, an immune responsein a subject infected, or suspected of having been infected, withpneumococcus is characterized. In some embodiments, an immune responsein a subject administered an immunogenic composition comprising apneumococcal antigen (e.g., an immunogenic composition described herein)is characterized. In some embodiments, an antibody response ischaracterized. In some embodiments, a B cell response is characterized.In some embodiments, a T cell response is characterized. In someembodiments, IFN-γ secretion by antigen-specific T cells ischaracterized. In some embodiments, a Th1 T cell response ischaracterized. In some embodiments, a Th17 T cell response ischaracterized, and in some embodiments, IL-17A secretion ischaracterized. In some embodiments, a cytotoxic T cell response ischaracterized. In some embodiments, both a T cell and a B cell responseare characterized. In some embodiments, an innate immune response ischaracterized.

The invention further provides methods of preparing compositionsincluding pneumococcual antigens, and antibodies that specifically bindto pneumococcal antigens as disclosed in Table 1.

Compositions and methods described herein can be used for theprophylaxis and/or treatment of any pneumococcus disease, disorder,and/or condition due to a pneumococcual infection. In some embodiments,an immunogenic composition described herein reduces risk of infectionby, and/or treats, alleviates, ameliorates, relieves, delays onset of,inhibits progression of, reduces severity of, and/or reduces incidenceof one or more symptoms or features of a pneumococcal disease, disorder,and/or condition. In some embodiments, the prophylaxis and/or treatmentof pneumococcal infection comprises administering a therapeuticallyeffective amount of an immunogenic composition comprising a novelpneumococcal antigen described herein to a subject in need thereof, insuch amounts and for such time as is necessary to achieve the desiredresult. In certain embodiments of the present invention a“therapeutically effective amount” of an inventive immunogeniccomposition is that amount effective for treating, alleviating,ameliorating, relieving, delaying onset of, inhibiting progression of,reducing severity of, and/or reducing incidence of one or more symptomsor features of pneumococcal infection.

In some embodiments, inventive prophylactic, prognostic and/ortherapeutic protocols involve administering a therapeutically effectiveamount of one or more immunogenic compositions comprising a novelpneumococcal antigen to a subject such that an immune response isstimulated in one or both of T cells and B cells. The present inventionalso provides novel immunogenic compositions comprising atherapeutically effective amount of one or more pneumococcal antigens(e.g., one or more of a polypeptide antigen selected from Table 1 orcombinations thereof) and one or more pharmaceutically acceptableexcipients. In some embodiments, the present invention provides forpharmaceutical compositions comprising an immunogenic composition asdescribed herein. In accordance with some embodiments, a method ofadministering a pharmaceutical composition comprising inventivecompositions to a subject (e.g. human, e.g., a child, adolescent, oryoung adult) in need thereof is provided.

In some embodiments, a therapeutically effective amount of animmunogenic composition is delivered to a patient and/or animal priorto, simultaneously with, and/or after diagnosis with a pneumococcaldisease, disorder, and/or condition. In some embodiments, a therapeuticamount of an inventive immunogenic composition is delivered to a patientand/or animal prior to, simultaneously with, and/or after onset ofsymptoms of a pneumococcal disease, disorder, and/or condition.

In some embodiments, immunogenic compositions of the present inventionare administered by any of a variety of routes, including oral,intramuscular, subcutaneous, transdermal, intradermal, rectal,intravaginal, mucosal, nasal, buccal, enteral, sublingual; byintratracheal instillation, bronchial instillation, and/or inhalation;and/or as an oral spray, nasal spray, and/or aerosol. In someembodiments, immunogenic compositions of the present invention areadministered by a variety of routes, including intravenous,intra-arterial, intramedullary, intrathecal, intraventricular,transdermal, intraperitoneal, topical (as by powders, ointments, creams,and/or drops), transdermal, or by intratracheal instillation.

In certain embodiments, an immunogenic composition may be administeredin combination with one or more additional therapeutic agents whichtreat the symptoms of pneumococcal infection (e.g., with an antibioticsuch as a beta-lactam antibiotic, an erythromycin, or a tetracycline).

Another aspect of the present invention relates to the use of theimmunogenic composition as disclosed herein to be administered to asubject to elicit an immune response in the subject. In someembodiments, the immune response is an antibody/B cell response, a CD4+T cell response (including Th1, Th2 and Th17 cells) and/or a CD8+ T-cellresponse. In some embodiments, at least one adjuvant is administered inconjunction with the immunogenic composition.

Another aspect of the present invention relates to a method for inducingan immune response in a subject to at least one antigen, comprisingadministering to the subject the immunogenic composition as disclosedherein.

Another aspect of the present invention relates to a method ofvaccinating an animal, e.g., a bird, a mammal or a human, against atleast one antigen comprising administering a vaccine compositioncomprising the immunogenic composition as disclosed herein.

In all aspects as disclosed herein, an animal or a subject can be ahuman. In some embodiments, the subject can be an agricultural ornon-domestic animal, or a domestic animal. In some embodiments, avaccine composition comprising the immunogenic composition as disclosedherein can be administered via subcutaneous, intranasal, oral,sublingual, vaginal, rectal, intradermal, intraperitoneal, intramuscular injection, or via skin-patch for transcutaneous immunization.

In all aspects as disclosed herein, an immune response is an antibody/Bcell response, a CD4+ T cell response (including Th1, Th2 and Th17responses) or a CD8+ T-cell response against protein/peptide antigen(s).In some embodiments, an immune response is an antibody/B cell responseagainst the polymer, e.g., a pneumococcal polysaccharide. In someembodiments, at least one adjuvant is administered in conjunction withthe immunogenic composition.

Another aspect of the present invention relates to the use of theimmunogenic composition as disclosed herein for use in a diagnostic forexposure to a pathogen or immunogenic agent.

Another aspect of the present invention relates to kits for preparing animmunogenic composition as disclosed herein. For example, such kits cancomprise any one or more of the following materials: a containercomprising a polymer, e.g., a polysaccharide, cross-linked with aplurality of first affinity molecules; and a container comprising acomplementary affinity molecule which associates with the first affinitymolecule, wherein the complementary affinity molecule associates with anantigen.

The invention provides a variety of kits comprising one or more of theimmunogenic compositions of the invention. For example, the inventionprovides a kit comprising an immunogenic composition comprising apneumococcual antigen, or a nucleic acid encoding the antigen, whereinthe antigen is selected from Table 1 or functional fragments orcombinations thereof; and instructions for use. A kit may comprisemultiple different pneumococcal antigens. A kit may comprise any of anumber of additional components or reagents in any combination.According to certain embodiments of the invention, a kit may include,for example, (i) a pneumococcal polypeptide antigen selected from Table1 or combinations thereof; (ii) an adjuvant; and (iii) instructions foradministering a composition including the pneumococcal antigen and theadjuvant to a subject in need thereof.

In some embodiments, the kit can comprise a container comprising anexpression vector for expressing a pneumococcal polypeptide antigenselected from Table 1, for example. In some embodiments, the vector canoptionally comprise a sequence for a linker peptide, wherein theexpression vector can expresses a pneumococcal polypeptide antigenselected from Table 1.

Provided herein also is a method of vaccinating a subject, e.g., amammal, e.g., a human with the immunogenic compositions as disclosedherein, the method comprising administering a vaccine composition asdisclosed herein to the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show that pneumococcus antigens provide protection againstcolonization. FIG. 1A shows decreased colony forming units (CFU) in anasal wash of mice challenged with serotype 6B pneumococcal strain aftertwice weekly immunization with SP0785 in the presence of the adjuvantcholera toxin (CT) as compared to CT alone. FIG. 1B shows decreasedcolony forming units (CFU) in a nasal wash of mice challenged withserotype 6B pneumococcal strain after twice weekly immunization withSP1500 in the presence of the adjuvant cholera toxin (CT) as compared toCT alone.

FIG. 2 shows pneumococcus antigens fused to Pdt (pneumococcalpneumolysoid PdT) protects against sepsis challenge and prevents againstinvasive infection and nasopharyngeal colonization of pneumococcus. FIG.2 is a survival curve of mice with sepsis showing that immunization witha fusion protein consisting of SP0785-PdT protected 80% mice againstsepsis, and fusion protein SP2145-PdT protected 90% of mice againstsepsis as compared to mice immunized with control mice (Alum only) orPdT alone (which protected only 30% of mice).

FIGS. 3A-3B show pneumococcal antigens fused to PdT (pneumococcalpneumolysoid PdT) and covalently conjugated to the Vi polysaccharidegenerates IL-17A and anti-Vi antibody; protecting against sepsischallenge and preventing invasive infection and nasopharyngealcolonization of pneumococcus. FIG. 3A shows high levels of IL-17A whenstimulated with whole cell vaccine (WCV) after the mice were immunizedwith SP0785-PdT-Vi conjugate or SP2145-PdT-Vi conjugate as compared tothe control Alum alone. FIG. 3B shows high levels of anti-Vi IgG in micewere immunized with SP0785-PdT-Vi conjugate or SP2145-PdT-Vi conjugate,as compared to Alum alone.

FIG. 4 shows pneumococcal antigens fused to Pdt (pneumococcalpneumolysoid PdT) and covalently conjugated to the Vi polysaccharideprotects against sepsis challenge and prevents against invasiveinfection and nasopharyngeal colonization of pneumococcus. FIG. 4 is asurvival curve of mice with sepsis showing that immunization with afusion protein consisting of SP2145-PdT-Vi or SP0785-PdT-Vi protected80% mice against sepsis as compared to mice immunized with control mice(Alum only) (which protected only 20% of mice).

FIG. 5 shows sera from immunized mice bind encapsulated pneumococcalstrain Tigr4. FIG. 5 shows results from a flow cytometric assay showingthat antibodies present in the serum from mice immunized with SP0785(black) can label the encapsulated pneumococcal strain Tigr4 as comparedto serum obtained from mice immunized with alum alone (grey). Thus, FIG.5 demonstrates that anti-SP0785 antibody is able to bind to encapsulatedpneumococcal strain.

FIGS. 6A-6B demonstrate protection against pneumococcal colonization bya Multiple Antigen Presenting System (MAPS) construct. MAPS complexeswere made using biotinylated type-1 pneumococcal polysaccharide attachedto a fusion protein consisting of rhizavidin and SP0785, SP1500, SP0435or PdT. C57/BL6 mice were immunized with a mixture of 3 MAPS complexescontaining SP0785, SP1500 and PdT, or a mixture of all 4 MAPS complexesdescribed above, on aluminum hydroxide, at a dosage of 6.7 μg of eachantigen. Control mice received either aluminum hydroxide alone (alum,negative control) or a whole cell vaccine in alum (as a positivecontrol). Immunization was given subcutaneously three times, two weeksapart. Blood was drawn after the third immunization and stimulated with10 μg/ml of S. pneumoniae whole cell antigen or 5 μg/ml of purifiedSP0785, SP1500 or PdT protein. Antigen-specific or whole cell (WCB)IL-17A production was measured 7 days post stimulation by ELISA (FIG.6A). One week after bleeding, mice were challenged with pneumococcal603B strain and bacterial colonization rate in the nose was determined10 days post challenge (FIG. 6B). Mice that received immunizations witheither 3 or 4 MAPS complexes were protected against pneumococcalcolonization.

FIG. 7 demonstrates protection against pneumococcal colonization by afusion protein of SP0785, SP1500 and PdT conjugated to Vi polysaccharideof Salmonella typhi. C57BL/6 mice were immunized with vaccinescontaining 5 μg of protein antigen adsorbed onto alum, subcutaneouslythree times, two weeks apart. Control mice received alum alone. Twoweeks after last immunization, mice were intranasally challenged withpneumococcal 603B strain and bacterial colonization rate in the nose wasdetermined by nasal wash. The mice that were immunized with the fusionconjugate were significantly protected against pneumococcal colonizationcompared to the mice in the control group.

FIG. 8 demonstrates that passive transfer of rabbit serum protects miceagainst pneumococcal challenge. New Zealand white rabbits were immunizedwith purified SP0785, SP1500 or PdT. An equal mixture of each serum fromrabbits pre-immunization vs. post immunization (in this case, 67 μleach) was given to groups of 10 mice via the intraperitoneal route. Micewere then intraperitoneally infected with a serotype 3 strain 24 hourslater and mice survival was monitored for 8 days. Group received postimmunization serum had 60% survival comparing to 0% survival in thepre-serum group, a difference that was highly statistically significant,attesting to the protective capacity of antibodies to these proteins forpneumococcal invasive disease.

DETAILED DESCRIPTION OF THE INVENTION

Streptococcus pneumoniae (S. pneumoniae) is a common cause of bacterialpneumonia, meningitis, otitis media, and bacteremia in children, theelderly, and immunodeficient individuals. S. pneumoniae can besubdivided into approximately 90 serotypes, based on the capsularpolysaccharide of the organism. However, disease is generally caused byapproximately 30 types of S. pneumoniae isolates. The World HealthOrganization estimates that there are one million deaths among childrendue to pneumococcal meningitis and sepsis each year, with 98% of thesedeaths occurring in developing countries. The emergence of pneumococcalstrains with antimicrobial resistance underscores the need for treatingand preventing pneumococcal infection by methods in addition toantimicrobials.

One aspect of the present invention encompasses the discovery of novelantigens for pneumococcus that elicit antigen specific immune responsesin mammals. Such novel antigens, and/or nucleic acids encoding theantigens, can be incorporated into immunogenic compositions andadministered to elicit immune responses, e.g., to provide protectionagainst pneumococcal colonization, invasive diseases, such as but notlimited to, sepsis, and diseases and disorders caused by pneumococcusorganisms. Such novel antigens and/or responses to novel antigens can bedetected to identify and/or characterize immune responses topneumococcal organisms.

Another aspect of the present invention provides immunogeniccompositions (e.g., vaccines) comprising at least one isolatedpneumococcus antigen selected from the pneumococcus proteins listed inTable 1, e.g., SP0010, SP0043, SP0079, SP0084, SP0092, SP0098, SP0106,SP0107, SP0127, SP0149, SP0191, SP0198, SP0249, SP0321, SP0346, SP0402,SP0453, SP0564, SP0582, SP0589, SP0601, SP0604, SP0617, SP0620, SP0629,SP0648, SP0659, SP0662, SP0664, SP0678, SP0724, SP0742, SP0757, SP0785,SP0787, SP0872, SP0878, SP0899, SP1002, SP1026, SP1032, SP1069, SP1154,SP1267, SP1376, SP1386, SP1404, SP1405, SP1419, SP1479, SP1500, SP1545,SP1560, SP1624, SP1652, SP1683, SP1826, SP1872, SP1891, SP1897, SP1942,SP1966, SP1967, SP1998, SP2048, SP2050, SP2083, SP2084, SP2088, SP2145,SP2151, SP2187, SP2192, SP2197, SP2207 and SP2218, or functionalfragments thereof, such as those shown in SEQ ID NO: 153-234. In someembodiments, an immunogenic composition comprising at least one isolatedpneumococcus antigen selected from the pneumococcus proteins of SP0785,SP1500, SP0346, SP1386, SP0084, SP1479 and SP2145 which protect againstpneumococcus and pneumococcal infection, such as sepsis.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

Definitions

The term “antigen” as used herein refers to a molecule (e.g., apolypeptide) that elicits a specific immune response. Antigen specificimmunological responses, also known as adaptive immune responses, aremediated by lymphocytes (e.g., T cells, B cells) that express antigenreceptors (e.g., T cell receptors, B cell receptors). In certainembodiments, an antigen is a T cell antigen, and elicits a cellularimmune response. In certain embodiments, an antigen is a B cell antigen,and elicits a humoral (i.e., antibody) response. In certain embodiments,an antigen is both a T cell antigen and a B cell antigen. As usedherein, the term “antigen” encompasses both a full-length polypeptide aswell as a portion of the polypeptide, that represent immunogenicfragments (i.e., fragments that elicit an antigen specific T cellresponse, B cell response, or both) of such complete polypeptides. Insome embodiments, antigen is a peptide epitope found within apolypeptide sequence (e.g., a peptide epitope bound by a MajorHistocompatibility Complex (MHC) molecule (e.g., MHC class I, or MHCclass II). Accordingly, peptides 5-15 amino acids in length, and longerpolypeptides, e.g., having 60, 70, 75, 80, 85, 90, 100, 150, 200 250, ormore amino acids, can be “antigens”. In an exemplary example, thepresent invention provides a SP0785 polypeptide antigen. In someembodiments, a SP0785 polypeptide antigen includes a full-length SP0785polypeptide amino acid sequence (e.g., a full-length SP0785 polypeptideof SEQ ID NO:34). In some embodiments, a SP0785 polypeptide antigenincludes a portion of a SP0785 polypeptide (e.g., a portion of theSP0785 polypeptide of SEQ ID NO:34, which portion includes at least 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, or400 contiguous amino acids of SEQ ID NO:34). In some embodiments, aportion of a SP0785 polypeptide corresponds to a protein having theamino acid sequence of SEQ ID NO: 190. In some embodiments, a SP0785polypeptide antigen contains one or more amino acid alterations (e.g.,deletion, substitution, and/or insertion) from a naturally-occurringwild-type SP0785 polypeptide sequence. For example, a SP0785 polypeptideantigen may contain an amino acid sequence that is at least 60% (e.g.,at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) identical to SEQ IDNO:34 or a portion thereof (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90,95, 100, 150, 200, 250, 300, 350, or 400 consecutive amino acids of thesequence shown in SEQ ID NO:34). Alternatively, a SP0785 polypeptideantigen may contain a portion (e.g., at least 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85,90, 95, 100, 150, 200, 250, 300, 350, or 400 consecutive amino acids) ofa sequence that is at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%,90%, 95%, or 98%) identical to SEQ ID NO:34 or SEQ ID NO: 190. SP0785polypeptide antigen is used as an example. This concept is applicable toother polypeptide antigen described herein including, but not limitedto, SP1500, SP0346, SP1386, SP0084, SP1479, and SP2145 polypeptideantigens, and any of those polypeptide antigens listed in Table 1.

The term “pneumococcal antigen” refers to an antigen that elicits anantigen specific immune response against any organism of thepneumococcal genus, such as a Streptococcus pneumonia organism, etc. Insome embodiments, a pneumococcal antigen elicits an antigen specificimmune response against S. pneumonia organisms of multiple species.Pneumococcal antigens include full-length polypeptides encoded bypneumococcal genes shown in Table 1 (SEQ ID NO: 1-76), as well asimmunogenic portions of the polypeptides shown in Table 2 (SEQ ID NOs:153-234).

The term “immunogenic composition” as used herein refers to acomposition that is capable of eliciting an immune response, such as anantibody or cellular immune response, when administered to a subject. Insome embodiments, an immunogenic composition includes a polypeptide orpeptide antigen. The immunogenic compositions of the present inventionmay or may not be immunoprotective or therapeutic. When the immunogeniccompositions of the present invention prevent, ameliorate, palliate oreliminate disease from the subject, then the immunogenic composition mayoptionally be referred to as a vaccine. As used herein, however, theterm immunogenic composition is not intended to be limited to vaccines.In some embodiments, an immunogenic composition includes a nucleic acidencoding a polypeptide or peptide antigen. An immunogenic compositioncan include molecules that induce an immune response against multipleantigens.

The term “antibody” as used herein refers to any immunoglobulin, whethernatural or wholly or partially synthetically produced. All derivativesthereof that maintain specific binding ability are also included in theterm. The term also covers any protein having a binding domain which ishomologous or largely homologous to an immunoglobulin-binding domain.Such proteins may be derived from natural sources, or partly or whollysynthetically produced. An antibody may be monoclonal or polyclonal. Anantibody may be a member of any immunoglobulin class, including any ofthe human classes: IgG, IgM, IgA, IgD, and IgE. As used herein, theterms “antibody fragment” or “characteristic portion of an antibody” areused interchangeably and refer to any derivative of an antibody which isless than full-length. In general, an antibody fragment retains at leasta significant portion of the full-length antibody's specific bindingability. Examples of antibody fragments include, but are not limited to,Fab, Fab′, F(ab′)2, scFv, Fv, dsFv diabody, and Fd fragments. Anantibody fragment may be produced by any means. For example, an antibodyfragment may be enzymatically or chemically produced by fragmentation ofan intact antibody and/or it may be recombinantly produced from a geneencoding the partial antibody sequence. Alternatively or additionally,an antibody fragment may be wholly or partially synthetically produced.An antibody fragment may optionally comprise a single chain antibodyfragment. Alternatively or additionally, an antibody fragment maycomprise multiple chains which are linked together, for example, bydisulfide linkages. An antibody fragment may optionally comprise amultimolecular complex. A functional antibody fragment will typicallycomprise at least about 50 amino acids and more typically will compriseat least about 200 amino acids.

The terms “Cytotoxic T Lymphocyte” or “CTL” refers to lymphocytes whichinduce death via apoptosis or other mechanisms in targeted cells. CTLsform antigen-specific conjugates with target cells via interaction ofTCRs with processed antigen (Ag) on target cell surfaces, resulting inapoptosis of the targeted cell. Apoptotic bodies are eliminated bymacrophages. The term “CTL response” is used to refer to the primaryimmune response mediated by CTL cells.

The term “cell mediated immunity” or “CMI” as used herein refers to animmune response that does not involve antibodies or complement butrather involves the activation of, for example, macrophages, naturalkiller cells (NK), antigen-specific cytotoxic T-lymphocytes (T-cells),T-helper cells, neutrophils, and the release of various cytokines inresponse to a target antigen. Stated another way, CMI refers to immunecells (such as T cells and other lymphocytes) which bind to the surfaceof other cells that display a target antigen (such as antigen presentingcells (APC)) and trigger a response. The response may involve eitherother lymphocytes and/or any of the other white blood cells (leukocytes)and the release of cytokines. Cellular immunity protects the body by:(i) activating antigen-specific cytotoxic T-lymphocytes (CTLs) that areable to destroy body cells displaying epitopes of foreign antigen ontheir surface, such as virus-infected cells and cells with intracellularbacteria; (2) activating macrophages and NK cells, enabling them todestroy intracellular pathogens; and (3) stimulating cells to secrete avariety of cytokines or chemokines that influence the function of othercells such as T cells, macrophages or neutrophils involved in adaptiveimmune responses and innate immune responses.

The term “immune cell” as used herein refers to any cell which canrelease a cytokine, chemokine or antibody in response to a direct orindirect antigenic stimulation. Included in the term “immune cells”herein are lymphocytes, including natural killer (NK) cells, T-cells(CD4+ and/or CD8+ cells), B-cells, macrophages; leukocytes; dendriticcells; mast cells; monocytes; and any other cell which is capable ofproducing a cytokine or chemokine molecule in response to direct orindirect antigen stimulation. Typically, an immune cell is a lymphocyte,for example a T-cell lymphocyte.

The term “cytokine” as used herein refers to a molecule released from animmune cell in response to stimulation with an antigen. Examples of suchcytokines include, but are not limited to: GM-CSF; IL-1α; IL-1β; IL-2;IL-3; IL-4; IL-5; IL-6; IL-7; IL-8; IL-10; IL-12; IL-17A, IL-17F orother members of the IL-17 family, IL-22, IL-23, IFN-γ; IFN-β; IFN-α;MIP-1α; MIP-1β; TGF-α; TNFα□ or TNFβ. The term “cytokine” does notinclude antibodies

The term “in vitro” as used herein refers to events that occur in anartificial environment, e.g., in a test tube or reaction vessel, in cellculture, etc., rather than within an organism (e.g., animal, plant,and/or microbe).

As used herein, the term “in vivo” refers to events that occur within anorganism (e.g., animal, plant, and/or microbe).

The term “isolated” as used herein, means that the isolated entity hasbeen separated from at least one component with which it was previouslyassociated. When most other components have been removed, the isolatedentity is “purified.” Isolation and/or purification and/or concentrationmay be performed using any techniques known in the art including, forexample, chromatography, fractionation, precipitation, or otherseparation.

The term “adjuvant” as used herein refers to any agent or entity whichincreases the antigenic response (e.g., immune response) by a cell or asubject to a target antigen. In some embodiments, an adjuvant is used toenhance an immune response to a peptide antigen administered to asubject. In some embodiments, an adjuvant is used to enhance an immuneresponse to an antigen encoded by a nucleic acid administered to asubject.

As used herein, the term “pathogen” refers to an organism or moleculethat causes a disease or disorder in a subject. For example, pathogensinclude but are not limited to viruses, fungi, bacteria, parasites andother infectious organisms or molecules therefrom, as well astaxonomically related macroscopic organisms within the categories algae,fungi, yeast and protozoa or the like.

As used herein, the term “prokaryotic pathogen” refers to a bacterialpathogen.

As used herein, the term “viral pathogen” refers to a virus that causesillness or disease, such as HIV.

As used herein, the term “parasitic pathogen” refers to a microorganismthat is parasitic, residing for an extended period inside a host cell orhost organism that gains benefits from the host and at the same timecauses illness or disease. A parasitic pathogen can be bacteria,viruses, fungi, and parasites, and protists.

The term “functional fragment” or “portion” as used in the context of afunctional fragment of an immunogen of protein “x” (e.g., anpneumococcal antigen or immunogen listed Table 1) refers to a fragmentof such a protein or peptide that mediates, effects or elicits acellular and/or humoral immune response as similar to the protein orpeptide from which it was derived (e.g. a fragment thereof).Accordingly, the term “functional” when used in conjunction with“derivative” or “variant” or “fragment” refers to a polypeptide whichpossess a biological activity that is substantially similar to abiological activity of the entity or molecule of which it is aderivative or variant or fragment thereof. By “substantially similar” inthis context is meant that at least 25%, at least 35%, at least 50% ofthe relevant or desired biological activity of a corresponding wild-typepeptide is retained. In the instance of a fragment of a pneumococcalantigen, such as for example, SP0785 (e.g., SEQ ID NO: 34), a functionalfragment of SEQ ID NO: 34 or SEQ ID NO: 190 would be a protein orpeptide comprising a portion of SEQ ID NO: 34 or SEQ ID NO: 190 whichretained an activity for eliciting an IL-17A response in splenocytes orhuman PMBC, and protect against colonization by pneumococcal organism;preferably the fragment of SEQ ID NO: 34 or SEQ ID NO: 190 retains atleast 25%, at least 35%, at least 50% at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 100% or even higher(i.e., the variant or derivative has greater activity than thewild-type), e.g., at least 110%, at least 120%, or more activity ascompared to the full length SEQ ID NO: 34 or SEQ ID NO: 190 to elicit anIL-17A response in splenocytes or human PMBC and protect againstcolonization by pneumococcus. Such functional fragments of theimmunogens (e.g. antigens) listed in Table 1 can be assessed by theassays as disclosed in the Examples, e.g., to assess if a functionalfragment elicits an IL-17A response in mouse splenocytes or human PBMCsin vitro as disclosed in Example 2, or protects colonization in vivo ina mouse colonization model, where mice are challenged with a serotype ofpneumococcal strain (e.g., serotypes 6B, 14F or 19F) after biweeklyimmunization with a pneumococcal antigen (or functional fragment), andassessing for presence of pneumococcal colonization in the nasopharynx,as disclosed in Example 3.

A “fragment” of an antigen or immunogen of Table 1 as that term is usedherein will be at least 15 amino acids in length, and can be, forexample, at least 16, at least 17, at least 18, at least 19, at least 20or at least 25 amino acids or greater inclusive.

The term “Cytotoxic T Lymphocyte” or “CTL” refers to lymphocytes whichinduce apoptosis in targeted cells. CTLs form antigen-specificconjugates with target cells via interaction of TCRs with processedantigen (Ag) on target cell surfaces, resulting in apoptosis of thetargeted cell. Apoptotic bodies are eliminated by macrophages. The term“CTL response” is used to refer to the primary immune response mediatedby CTL cells.

The term “cell mediated immunity” or “CMI” as used herein refers to animmune response that does not involve antibodies or complement butrather involves the activation of macrophages, natural killer cells(NK), antigen-specific cytotoxic T-lymphocytes (T-cells), and therelease of various cytokines in response to a target antigen. Statedanother way, CMI refers to immune cells (such as T cells andlymphocytes) which bind to the surface of other cells that display theantigen (such as antigen presenting cells (APS)) and trigger a response.The response can involve either other lymphocytes and/or any of theother white blood cells (leukocytes) and the release of cytokines.Accordingly, cell-mediated immunity (CMI) is an immune response thatdoes not involve antibodies but rather involves the activation ofmacrophages and NK-cells, the production of antigen-specific cytotoxicT-lymphocytes, and the release of various cytokines in response to anantigen. Cellular immunity protects the body by: (i) activatingantigen-specific cytotoxic T-lymphocytes (CTLs) that are able to destroybody cells displaying epitopes of foreign antigen on their surface, suchas virus-infected cells, cells with intracellular bacteria, and cancercells displaying tumor antigens; (2) activating macrophages and NKcells, enabling them to destroy intracellular pathogens; and (3)stimulating cells to secrete a variety of cytokines that influence thefunction of other cells involved in adaptive immune responses and innateimmune responses. Without wishing to be bound by theory and by way ofbackground, the immune system was separated into two branches: humoralimmunity, for which the protective function of immunization could befound in the humor (cell-free bodily fluid or serum) and cellularimmunity, for which the protective function of immunization wasassociated with cells.

The term “immune cell” as used herein refers to any cell which canrelease a cytokine in response to a direct or indirect antigenicstimulation. Included in the term “immune cells” herein are lymphocytes,including natural killer (NK) cells, T-cells (CD4+ and/or CD8+ cells),B-cells, macrophages and monocytes, Th cells; Th1 cells; Th2 cells; Tccells; stromal cells; endothelial cells; leukocytes; dendritic cells;macrophages; mast cells and monocytes and any other cell which iscapable of producing a cytokine molecule in response to direct orindirect antigen stimulation. Typically, an immune cell is a lymphocyte,for example a T-cell lymphocyte.

The term “nucleic acid” as used herein, in its broadest sense, refers toany compound and/or substance that is or can be incorporated into anoligonucleotide chain. In some embodiments, a nucleic acid is a compoundand/or substance that is or can be incorporated into an oligonucleotidechain via a phosphodiester linkage. As used herein, the terms“oligonucleotide” and “polynucleotide” can be used interchangeably. Insome embodiments, “nucleic acid” encompasses RNA as well as singleand/or double-stranded DNA and/or cDNA. Furthermore, the terms “nucleicacid,” “DNA,” “RNA,” and/or similar terms include nucleic acid analogs,i.e. analogs having other than a phosphodiester backbone. The term“nucleotide sequence encoding an amino acid sequence” includes allnucleotide sequences that are degenerate versions of each other and/orencode the same amino acid sequence. Nucleic acids can be purified fromnatural sources, produced using recombinant expression systems andoptionally purified, chemically synthesized, etc. Where appropriate,e.g., in the case of chemically synthesized molecules, nucleic acids cancomprise nucleoside analogs such as analogs having chemically modifiedbases or sugars, backbone modifications, etc. A nucleic acid sequence ispresented in the 5′ to 3′ direction unless otherwise indicated.

The term “polypeptide”, as used herein, generally has its art-recognizedmeaning of a polymer of at least three amino acids. However, the term isalso used to refer to specific classes of antigen polypeptides, such as,for example, SP0785 polypeptides, SP1500 polypeptides, SP0346polypeptides, SP1386 polypeptides, SP0084 polypeptides, SP1479polypeptides and SP2145 polypeptides. For each such class, the presentspecification provides several examples of known sequences of suchpolypeptides. Those of ordinary skill in the art will appreciate,however, that the term “polypeptide”, as used herein to refer to“polypeptide antigen”, is intended to be sufficiently general as toencompass not only polypeptides having a sequence recited herein, butalso to encompass polypeptides having a variation of the sequence thatelicits an antigen-specific response to the polypeptide. For example, a“SP0785 polypeptide” includes the SP0785 polypeptide shown in SEQ IDNO:34, as well as polypeptides that have variations of a SEQ ID NO:34sequence, such as for example, a fragment of SP0785 as shown in SEQ IDNO: 190, and that maintain the ability to elicit an antigen-specificresponse to a polypeptide of SEQ ID NO:34. Those of ordinary skill inthe art understand that protein sequences generally tolerate somesubstitution without destroying immunogenicity and antigen specificity.Thus, any polypeptide that retains immunogenicity and shares at leastabout 30-40% overall sequence identity, often greater than about 50%,60%, 70%, or 80%, and further usually including at least one region ofmuch higher identity, often greater than 90% or even 95%, 96%, 97%, 98%,or 99% in one or more highly conserved regions, usually encompassing atleast 3-4 and often up to 20 or more amino acids, with anotherpolypeptide of the same class, is encompassed within the relevant term“polypeptide” as used herein. Other regions of similarity and/oridentity can be determined by those of ordinary skill in the art byanalysis of the sequences of various polypeptides presented herein. Seethe definition of Antigen. It will be appreciated that proteins orpolypeptides often contain amino acids other than the 20 amino acidscommonly referred to as the 20 naturally occurring amino acids, and thatmany amino acids, including the terminal amino acids, can be modified ina given polypeptide, either by natural processes such as glycosylationand other post-translational modifications, or by chemical modificationtechniques which are well known in the art. Known modifications whichcan be present in polypeptides of the present invention include, but arenot limited to, acetylation, acylation, ADP-ribosylation, amidation,covalent attachment of flavin, covalent attachment of a heme moiety,covalent attachment of a polynucleotide or polynucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutamate, formulation,gamma-carboxylation, glycation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination.

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977.BLAST is used, with the parameters described herein, to determinepercent sequence identity for the nucleic acids and proteins of thepresent disclosure. Software for performing BLAST analysis is publiclyavailable through the National Center for Biotechnology Information(available at the following internet address: ncbi.nlm.nih.gov). Thisalgorithm involves first identifying high scoring sequence pairs (HSPs)by identifying short words of length W in the query sequence, whicheither match or satisfy some positive-valued threshold score T whenaligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are extendedin both directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA, 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA, 90:5873-5787, 1993). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

The term “subject” is used interchangeably herein with “patient” or“individual” refers to any organism to which a composition of thisinvention may be administered, e.g., for experimental, diagnostic,and/or therapeutic purposes. Typical subjects include mammals such asmice, rats, rabbits, non-human primates, domestic animals and humans. Insome embodiments, the term “subject” refers to any animal in which it isuseful to elicit an immune response. The subject can be a wild,domestic, commercial or companion animal such as a bird or mammal. Thesubject can be a human. Although in one embodiment of the invention itis contemplated that the immunogenic compositions as disclosed hereincan also be suitable for the therapeutic or preventative treatment inhumans, it is also applicable to warm-blooded vertebrates, e.g.,mammals, such as non-human primates, (particularly higher primates),sheep, dog, rodent (e.g., mouse or rat), guinea pig, goat, pig, cat,rabbits, cows, and non-mammals such as chickens, ducks, or turkeys. Inanother embodiment, the subject is a wild animal, for example a birdsuch as for the diagnosis of avian flu. In some embodiments, the subjectis an experimental animal or animal substitute as a disease model. Thesubject may be a subject in need of veterinary treatment, whereeliciting an immune response to an antigen is useful to prevent adisease and/or to control the spread of a disease, for example SIV,STL1, SFV, or in the case of live-stock, hoof and mouth disease, or inthe case of birds Marek's disease or avian influenza, and other suchdiseases.

The term “recombinant” when used to describe a nucleic acid molecule,means a polynucleotide of genomic, cDNA, viral, semisynthetic, and/orsynthetic origin, which, by virtue of its origin or manipulation, is notassociated with all or a portion of the polynucleotide sequences withwhich it is associated in nature. The term recombinant as used withrespect to a peptide, polypeptide, protein, or recombinant fusionprotein, means a polypeptide produced by expression from a recombinantpolynucleotide. The term recombinant as used with respect to a host cellmeans a host cell into which a recombinant polynucleotide has beenintroduced. Recombinant is also used herein to refer to, with referenceto material (e.g., a cell, a nucleic acid, a protein, or a vector) thatthe material has been modified by the introduction of a heterologousmaterial (e.g., a cell, a nucleic acid, a protein, or a vector).

The term “vectors” refers to a nucleic acid molecule capable oftransporting or mediating expression of a heterologous nucleic acid towhich it has been linked to a host cell; a plasmid is a species of thegenus encompassed by the term “vector.” The term “vector” typicallyrefers to a nucleic acid sequence containing an origin of replicationand other entities necessary for replication and/or maintenance in ahost cell. Vectors capable of directing the expression of genes and/ornucleic acid sequence to which they are operatively linked are referredto herein as “expression vectors”. In general, expression vectors ofutility are often in the form of “plasmids” which refer to circulardouble stranded DNA molecules which, in their vector form are not boundto the chromosome, and typically comprise entities for stable ortransient expression or the encoded DNA. Other expression vectors thatcan be used in the methods as disclosed herein include, but are notlimited to plasmids, episomes, bacterial artificial chromosomes, yeastartificial chromosomes, bacteriophages or viral vectors, and suchvectors can integrate into the host's genome or replicate autonomouslyin the particular cell. A vector can be a DNA or RNA vector. Other formsof expression vectors known by those skilled in the art which serve theequivalent functions can also be used, for example self replicatingextrachromosomal vectors or vectors which integrates into a host genome.Preferred vectors are those capable of autonomous replication and/orexpression of nucleic acids to which they are linked.

An individual who is “suffering from” a disease, disorder, and/orcondition has been diagnosed with or displays one or more symptoms ofthe disease, disorder, and/or condition.

An individual who is “susceptible to” a disease, disorder, and/orcondition has not been diagnosed with and/or may not exhibit symptoms ofthe disease, disorder, and/or condition. In some embodiments, a disease,disorder, and/or condition is associated with a pneumococcal infection.Without wishing to be bound to theory, S. pneumoniae is responsible for15-50% of all episodes of community-acquired pneumonia, 30-50% of allcases of acute otitis media and a significant proportion of bacteremiaand bacterial meningitis. In some embodiments, an individual who issusceptible to a pneumococcal infection may be exposed to a pneumococcus(e.g., by ingestion, inhalation, physical contact, etc.). In someembodiments, an individual who is susceptible to a pneumococcalinfection may be exposed to an individual who is infected with themicrobe. In some embodiments, an individual who is susceptible to apneumococcal infection is one who is in a location where the microbe isprevalent (e.g., one who is traveling to a location where the microbe isprevalent). In some embodiments, an individual who is susceptible to apneumococcal infection is susceptible due to young age (e.g., a child,adolescent, or young adult). In some embodiments, a subject who issusceptible is to pneumococcal infection is a subject with Job'ssyndrome (subject lacking Th-17 cell-mediated response) or aaggamaglobunemic subject (a subject lacking antibody-mediated response).In some embodiments, a subject who is susceptible is a subject whoseimmune system is compromised, such as those living with HIV, or has anauto-immune disease, or has influenza. In some embodiments, a subjectwho is susceptible to a pneumococcal infection is a subject who hasother risk factors, such as, but not limited to smoking, injection druguse, Hepatitis C, and COPD. In some embodiments, an individual who issusceptible to a disease, disorder, and/or condition will develop thedisease, disorder, and/or condition. In some embodiments, an individualwho is susceptible to a disease, disorder, and/or condition will notdevelop the disease, disorder, and/or condition.

The term “therapeutically effective amount” as used herein means anamount of a therapeutic, prophylactic, and/or diagnostic agent (e.g.,inventive immunogenic composition) that is sufficient, when administeredto a subject suffering from or susceptible to a disease, disorder,and/or condition, to treat, alleviate, ameliorate, relieve, alleviatesymptoms of, prevent, delay onset of, inhibit progression of, reduceseverity of, and/or reduce incidence of the disease, disorder, and/orcondition.

The term “therapeutic agent” as used herein refers to any agent that,when administered to a subject, has a therapeutic, prophylactic, and/ordiagnostic effect and/or elicits a desired biological and/orpharmacological effect.

The term “treating” as used herein refers to the term “treating” refersto partially or completely alleviating, ameliorating, relieving,delaying onset of, inhibiting progression of, reducing severity of,and/or reducing incidence of one or more symptoms or features of aparticular disease, disorder, and/or condition. For example, “treating”a microbial infection may refer to inhibiting survival, growth, and/orspread of the microbe. Treatment may be administered to a subject whodoes not exhibit signs of a disease, disorder, and/or condition and/orto a subject who exhibits only early signs of a disease, disorder,and/or condition for the purpose of decreasing the risk of developingpathology associated with the disease, disorder, and/or condition. Insome embodiments, treatment comprises delivery of an immunogeniccomposition (e.g., a vaccine) to a subject.

The term “vaccine” as used herein refers to an entity comprising atleast one immunogenic component (e.g., an immunogenic component whichincludes a peptide or protein, and/or an immunogenic component whichincludes a nucleic acid). In certain embodiments, a vaccine includes atleast two immunogenic components. In some embodiments, a vaccine iscapable of stimulating an immune response of both T cells and B cells.In some embodiments, any assay available in the art may be used todetermine whether T cells and/or B cells have been stimulated. In someembodiments, T cell stimulation may be assayed by monitoringantigen-induced production of cytokines, antigen-induced proliferationof T cells, and/or antigen-induced changes in protein expression. Insome embodiments, B cell stimulation may be assayed by monitoringantibody titers, antibody affinities, antibody performance inneutralization assays, class-switch recombination, affinity maturationof antigen-specific antibodies, development of memory B cells,development of long-lived plasma cells that can produce large amounts ofhigh-affinity antibodies for extended periods of time, germinal centerreactions, and/or antibody performance in neutralization assays. In someembodiments, a vaccine further includes at least one adjuvant that canhelp stimulate an immune response in T cells and/or B cells.

The term “pharmaceutically acceptable” refers to compounds andcompositions which may be administered to mammals without unduetoxicity. The term “pharmaceutically acceptable carriers” excludestissue culture medium. Exemplary pharmaceutically acceptable saltsinclude but are not limited to mineral acid salts such ashydrochlorides, hydrobromides, phosphates, sulfates, and the like, andthe salts of organic acids such as acetates, propionates, malonates,benzoates, and the like. Pharmaceutically acceptable carriers are wellknown in the art.

The term “wild-type” as used herein refers to the typical or the mostcommon form existing in nature or as it normally exists in vivo.

The term “mutant” refers to an organism or cell with any change in itsgenetic material, in particular a change (i.e., deletion, substitution,addition, or alteration) relative to a wild-type polynucleotide sequenceor any change relative to a wild-type protein sequence. The term“variant” may be used interchangeably with “mutant”. Although it isoften assumed that a change in the genetic material results in a changeof the function of the protein, the terms “mutant” and “variant” referto a change in the sequence of a wild-type protein regardless of whetherthat change alters the function of the protein (e.g., increases,decreases, imparts a new function), or whether that change has no effecton the function of the protein (e.g., the mutation or variation issilent).

The term “reduced” or “reduce” or “decrease” as used herein generallymeans a decrease by a statistically significant amount relative to areference. For avoidance of doubt, “reduced” means statisticallysignificant decrease of at least 10% as compared to a reference level,for example a decrease by at least 20%, at least 30%, at least 40%, atleast t 50%, or least 60%, or least 70%, or least 80%, at least 90% ormore, up to and including a 100% decrease (i.e., absent level ascompared to a reference sample), or any decrease between 10-100% ascompared to a reference level, as that term is defined herein.

The term “low” as used herein generally means lower by a staticallysignificant amount; for the avoidance of doubt, “low” means astatistically significant value at least 10% lower than a referencelevel, for example a value at least 20% lower than a reference level, atleast 30% lower than a reference level, at least 40% lower than areference level, at least 50% lower than a reference level, at least 60%lower than a reference level, at least 70% lower than a reference level,at least 80% lower than a reference level, at least 90% lower than areference level, up to and including 100% lower than a reference level(i.e., absent level as compared to a reference sample).

The terms “increased” or “increase” as used herein generally mean anincrease by a statically significant amount; such as a statisticallysignificant increase of at least 10% as compared to a reference level,including an increase of at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 100% or more, inclusive, including, for example at least 2 fold,at least 3-fold, at least 4-fold, at least 5-fold, at least 10-foldincrease or greater as compared to a reference level, as that term isdefined herein.

The term “high” as used herein generally means a higher by a staticallysignificant amount relative to a reference; such as a statisticallysignificant value at least 10% higher than a reference level, forexample at least 20% higher, at least 30% higher, at least 40% higher,at least 50% higher, at least 60% higher, at least 70% higher, at least80% higher, at least 90% higher, at least 100% higher, inclusive, suchas at least 2-fold higher, at least 3-fold higher, at least 4-foldhigher, at least 5-fold higher, at least 10-fold higher or more, ascompared to a reference level.

As used herein and in the claims, the singular forms include the pluralreference and vice versa unless the context clearly indicates otherwise.Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.”

As used herein, the terms “approximately” or “about” in reference to anumber are generally taken to include numbers that fall within a rangeof 5%, 10%, 15%, or 20% in either direction (greater than or less than)of the number unless otherwise stated or otherwise evident from thecontext (except where such number would be less than 0% or exceed 100%of a possible value).

As used herein, the term “comprising” means that other elements can alsobe present in addition to the defined elements presented. The use of“comprising” indicates inclusion rather than limitation.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment of the invention.

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as those commonly understood to one of ordinaryskill in the art to which this invention pertains. Although any knownmethods, devices, and materials may be used in the practice or testingof the invention, the methods, devices, and materials in this regard aredescribed herein.

Pneumococcal Antigens

The inventors have previously demonstrated that one mechanism ofprotection against pneumococcal colonization was using a WCV (whole cellvaccine) that confers protection against both colonization and invasivedisease in mice. (Malley et al., 69 Infect. Immun. 4870-73 (2001);Malley et al., 74 Infect. Immun. 4290-92 (2004).). It was previouslydemonstrated that protection against colonization following immunizationwith WCV (whole cell vaccine) is antibody-independent and dependent onCD4+ T cells (Malley et al., 102 P.N.A.S. USA 102, 4848-53 (2005);Trzcinski et al., 73 Infect. Immun. 7043-46 (2005)). The effector T cellis the CD4+ TH17 cell, with neutralization of IL-17A response with ananti IL-17A antibody diminished the protection by the WCV, and IL-17Areceptor knockout mice are not protected by the WCV. In contrast,IFN-gamma or IL-4 deficient mice (which are skewed away from TH1 or TH2responses, respectively) are fully protected (Lu et al., 4 PLoSPathogens. e1000159 (2008)). Rats and mice immunized with the WCV arereported to be significantly protected against pneumococcal sepsis intwo pneumonia models (Malley et al., 2001).

Using bioinformatics analysis as disclosed herein, the inventors haveidentified specific pneumococcal antigens which are listed in Table 1which protect against pneumococcal colonization and infection, such asagainst pneumococcal sepsis infection. These pneumococcal antigenslisted in Table 1 can be administered as vaccines, in the presence orabsence of an adjuvant, to elicit a systemic IL-17A response and reduceor protect against intranasal pneumococcal colonization. In someembodiments, a pneumococcal antigen of any listed in Table 1 can beadministered as mucosal vaccines to protect against intranasalpneumococcal colonization as well as pneumococcal infection, such assepsis.

TABLE 1 Table 1 lists the amino acid sequence identification numbers ofthe pneumococcal immunogens. The amino acid sequences and nucleotidesequences of the pneumococcal antigens are available on world-wide website: “xbase.ac.uk/genome/streptococcus-pneumoniae-tigr4/NC_003028/features?page=1”, which is incorporated herein in itsentirety by reference. Protein DNA Immunogenic Pneumococcal SEQ SEQGenBank fragments of the antigen Predicted ID ID Gene Accessionpneumococcal name functions NO: NO: ID NO. No antigen SP0010hypothetical protein 1 77 15899959 NP_344563.1 SP0010 (23-end) (SEQ IDNO: 153) SP0043 competence factor 2 78 15899988 NP_344592.1 SP0043(42-end) transport protein (SEQ ID NO: 154) ComB SP0079 potassium uptake3 79 15900024 NP_344628.1 SP0079 (24-end) protein, Trk family (SEQ IDNO: 155) SP0084 histidine kinase (EC 4 80 15900028 NP_344632.1 SP0084(110-end) 2.7.13.3) (IMGterm) (SEQ ID NO: 156) SP0092 carbohydrate ABC 581 15900035 NP_344639.1 SP0092 (30-end) transporter substrate- (SEQ IDNO: 157) binding protein, CUT1 family (TC 3.A.1.1.-) (IMGterm) SP0098hypothetical protein 6 82 15900041 NP_344645.1 SP0098 (30-end) (SEQ IDNO: 158) SP0106 L-serine ammonia- 7 83 15900049 NP_344653.1 SP0106(29-end) lyase (EC 4.3.1.17) (SEQ ID NO: 159) (IMGterm) SP0107 LysMdomain protein 8 84 15900069 NP_344673.1 SP0107 (30-end) (SEQ ID NO:160) SP0127 hypothetical protein 9 85 15900069 NP_344673.1 SP0127(26-end) (SEQ ID NO: 161) SP0149 lipoprotein 10 86 15900087 NP_344691.1SP0149 (25-end) (SEQ ID NO: 162) SP0191 hypothetical protein 11 8715900128 NP_344732.1 SP0191 (26-end) (SEQ ID NO: 163) SP0198hypothetical protein 12 88 15900134 NP_344738.1 SP0198 (45-end) (SEQ IDNO: 164) SP0249 PTS system, IIB 13 89 15900184 NP_344788.1 SP0249(26-end) component (SEQ ID NO: 165) SP0321 PTS system, IIA 14 9015900253 NP_344857.1 SP0321 (1-end) component (SEQ ID NO: 14) SP0346capsular 15 91 15900275 NP_344879.1 SP0346 (98-end) polysaccharide (SEQID NO: 166) biosynthesis protein Cps4A SP0402 signal peptidase. 16 9215900321 NP_344925.1 SP0402 (29-end) Serine peptidase. (SEQ ID NO: 167)MEROPS family S26A (IMGterm) SP0453 amino acid ABC 17 93 15900370NP_344974.1 SP0453 (25-298) transporter substrate- (SEQ ID NO: 168)binding protein, PAAT family (TC 3.A.1.3.-)/amino acid ABC transportermembrane protein, PAAT family (TC 3.A.1.3.-) (IMGterm) SP0564hypothetical protein 18 94 15900476 NP_345080.1 SP0564 (21-end) (SEQ IDNO: 169) SP0582 hypothetical protein 19 95 15900492 NP_345096.1 SP0582(92-end) (SEQ ID NO: 170) SP0589 serine O- 20 96 15900498 NP_345102.1SP0589 (36-end) acetyltransferase (EC (SEQ ID NO: 171) 2.3.1.30)(IMGterm) SP0601 transmembrane 21 97 15900509 NP_345113.1 SP0601(36-297) protein Vexp3 (SEQ ID NO: 172) SP0604 sensor histidine 22 9815900512 NP_345116.1 SP0604 (223-end) kinase VncS (SEQ ID NO: 173)SP0617 hypothetical protein 23 99 15900525 NP_345129.1 SP0617 (44-end)(SEQ ID NO: 174) SP0620 amino acid ABC 24 100 15900528 NP_345132.1SP0620 (27-end) transporter substrate- (SEQ ID NO: 175) binding protein,PAAT family (TC 3.A.1.3.-) (IMGterm) SP0629 D-Ala-D-Ala 25 101 15900536NP_345140.1 SP0629 (21-end) carboxypeptidase. (SEQ ID NO: 176)Metallopeptidase. MEROPS family M15B (IMGterm) SP0648 beta-galactosidase26 102 15900551 NP_345155.1 SP0648 (40-776) (EC:3.2.1.23) (SEQ ID NO:177), (IMGterm) SP0648 (777-1676) (SEQ ID NO: 178), SP0648 (1677-end)(SEQ ID NO: 179) SP0659 thioredoxin family 27 103 15900560 NP_345164.1SP0659 (28-end) protein (SEQ ID NO: 180) SP0662 sensor histidine 28 10415900563 NP_345167.1 SP0662 (29-276) kinase, putative (SEQ ID NO: 181),SP0662 (300-end) (SEQ ID NO: 182) SP0664 zinc metalloprotease 29 10515900565 NP_345169.1 SP0664 (103-629) ZmpB (SEQ ID NO: 183), SP0664(630-1200) (SEQ ID NO: 184), SP0664 (1201-end) (SEQ ID NO: 185) SP0678hypothetical protein 30 106 15900579 NP_345183.1 SP0678 (23-end) (SEQ IDNO: 186) SP0724 hydroxyethylthiazole 31 107 15900621 NP_345225.1 SP0724(35-end) kinase, putative (SEQ ID NO: 187) SP0742 hypothetical protein32 108 15900637 NP_345241.1 SP0742 (43-end) (SEQ ID NO: 188) SP0757 celldivision protein 33 109 15900651 NP_345255.1 SP0757 (44-451) FtsX(IMGterm) (SEQ ID NO: 189) SP0785 hypothetical protein 34 110 15900678NP_345282.1 SP0785 (33-end) (SEQ ID NO: 190) SP0787 hypothetical protein35 111 15900680 NP_345284.1 SP0787 (43-290) (SEQ ID NO: 191) SP0872 D,D-36 112 15900755 NP_345359.1 SP0872 (30-end) carboxypeptidase (SEQ ID NO:192) PBP3. Serine peptidase. MEROPS family S11 (IMGterm) SP0878 SpoEfamily protein 37 113 15900761 NP_345365.1 SP0878 (245-end) (SEQ ID NO:193) SP0899 hypothetical protein 38 114 15900781 NP_345385.1 SP0899(31-end) (SEQ ID NO: 194) SP1002 adhesion lipoprotein 39 115 15900875NP_345479.1 SP1002 (22-end) (SEQ ID NO: 195) SP1026 hypothetical protein40 116 15900897 NP_345501.1 SP1026 (24-end) (SEQ ID NO: 196) SP1032iron-compound ABC 41 117 15900903 NP_345507.1 SP1032 (22-end)transporter, iron (SEQ ID NO: 197) compound-binding protein SP1069hypothetical protein 42 118 15900938 NP_345542.1 SP1069 (34-end) (SEQ IDNO: 198) SP1154 IgA1-specific 43 119 15901019 NP_345623.1 SP1154(155-694) metallopeptidase. (SEQ ID NO: 199), Metallo peptidase. SP1154(695-1374) MEROPS family (SEQ ID NO: 200), M26 (IMGterm) SP1154(1375-end) (SEQ ID NO: 201) SP1267 licC protein 44 120 15901127NP_345731.1 SP1267 (25-end) (SEQ ID NO: 202) SP1376 shikimate 45 12115901230 NP_345834.1 SP1376 (32-end) dehydrogenase (SEQ ID NO: 203) (EC1.1.1.25) (IMGterm) SP1386 spermidine/putrescine 46 122 15901240NP_345844.1 SP1386 (33-end) ABC transporter, (SEQ ID NO: 204)spermidine/putrescine- binding protein SP1404 hypothetical protein 47123 15901258 NP_345862.1 SP1404 (31-end) (SEQ ID NO: 205) SP1405transcriptional 48 124 15901259 NP_345863.1 SP1405 (19-end) regulatorSpx (SEQ ID NO: 206) SP1419 acetyltransferase, 49 125 15901272NP_345876.1 SP1419 (27-end) GNAT family (SEQ ID NO: 207) SP1479peptidoglycan N- 50 126 15901329 NP_345933.1 SP1479 (40-end)acetylglucosamine (SEQ ID NO: 208) deacetylase A SP1500 amino acid ABC51 127 15901347 NP_345951.1 SP1500 (27-end) transporter substrate- (SEQID NO: 209) binding protein , PAAT family (TC 3.A.1.3.-) (IMGterm)SP1545 hypothetical protein 52 128 15901388 NP_345992.1 SP1545 (29-end)(SEQ ID NO: 210) SP1560 hypothetical protein 53 129 15901403 NP_346007.1SP1560 (28-end) (SEQ ID NO: 211) SP1624 1-acyl-sn-glycerol- 54 13015901460 NP_346064.1 SP1624 (1-217) 3-phosphate (SEQ ID NO: 212)acyltransferase (EC 2.3.1.51) (IMGterm) SP1652 hypothetical protein 55131 15901487 NP_346091.1 SP1652 (62-397) (SEQ ID NO: 213) SP1683carbohydrate ABC 56 132 15901518 NP_346122.1 SP1683 (65-end) transportersubstrate- (SEQ ID NO: 214) binding protein, CUT1 family (TC 3.A.1.1.-)(IMGterm) SP1826 ABC transporter, 57 133 15901655 NP_346259.1 SP1826(36-end) substrate-binding (SEQ ID NO: 215) protein SP1872 iron-compoundABC 58 134 15901700 NP_346304.1 SP1872 (40-end) transporter, iron- (SEQID NO: 216) compound-binding protein SP1891 oligopeptide ABC 59 13515901718 NP_346322.1 SP1891 (40-end) transporter, (SEQ ID NO: 217)oligopeptide-binding protein AmiA SP1897 multiple sugar- 60 136 15901724NP_346328.1 SP1897 (30-end) binding protein (SEQ ID NO: 218) (IMGterm)SP1942 transcriptional 61 137 15901766 NP_346370.1 SP1942 (37-end)regulator, putative (SEQ ID NO: 219) SP1966 UDP-N- 62 138 15901789NP_346393.1 SP1966 (25-end) acetylglucosamine 1- (SEQ ID NO: 220)carboxyvinyltransferase (EC 2.5.1.7) (IMGterm) SP1967 hypotheticalprotein 63 139 15901790 NP_346394.1 SP1967 (30-end); (SEQ ID NO: 221)SP1998 L-asparaginase 64 140 15901821 NP_346425.1 SP1998 (51-end) (EC3.5.1.1) (SEQ ID NO: 222) (IMGterm) SP2048 hypothetical protein 65 14115901868 NP_346472.1 SP2048 (40-end) (SEQ ID NO: 223) SP2050 competenceprotein 66 142 15901870 NP_346474.1 SP2050 (35-end) CglD (SEQ ID NO:224) SP2083 sensor histidine 67 143 15901899 NP_346503.1 SP2083(192-end) kinase PnpS (SEQ ID NO: 225) SP2084 phosphate ABC 68 14415901900 NP_346504.1 SP2084 (30-end) transporter substrate- (SEQ ID NO:226) binding protein, PhoT family (TC 3.A.1.7.1) (IMGterm) SP2088phosphate uptake 69 145 15901904 NP_346508.1 SP2088 (30-end); regulator,PhoU (SEQ ID NO: 227) (IMGterm) SP2145 antigen, cell wall 70 14615901958 NP_346562.1 SP2145 (1-end) surface anchor family (SEQ ID NO:70) SP2151 carbamate kinase 71 147 15901963 NP_346567.1 SP2151 (25-end)(EC 2.7.2.2) (SEQ ID NO: 228) (IMGterm) SP2187 hypothetical protein 72148 15901994 NP_346598.1 SP2187 (32-end); (SEQ ID NO: 229) SP2192 sensorhistidine 73 149 15901999 NP_346603.1 SP2192 (224-end) kinase (SEQ IDNO: 230) SP2197 ABC transporter, 74 150 15902004 NP 346608.1 SP2197(30-end) substrate-binding (SEQ ID NO: 231) protein, putative SP2207competence protein 75 151 15902014 NP_346618.1 SP2207 (30-end) ComF,putative (SEQ ID NO: 232) SP2218 rod shape- 76 152 15902022 NP_346626.1SP2218 (106-end) determining (SEQ ID NO: 234) protein MreC (IMGterm)

In some cases, the other appropriate S. pneumoniae antigen is at leastat least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to thecorresponding wild-type S. pneumoniae protein disclosed in Table 1.Sequences of the above-mentioned polypeptides, and nucleic acids thatencode them, are known; see, for example, the S. pneumoniae ATCC 700669complete genome sequence under GenBank accession number FM211187.1 andlinked polypeptide sequences therein.

In addition to those nucleic acids and polypeptides described in Table 1above, this application also provides immunogenic compositions thatinclude one or more of the polypeptides or genes listed in Table 2, orvariants or fragments thereof as described herein. The DNA and proteinsequence of each gene and protein may be found by searching for theLocus Tag in the publicly available database, Entrez Gene, as describedabove.

In one aspect of the present invention provides immunogenic compositions(e.g., vaccines) comprising at least one isolated pneumococcus antigenselected from the pneumococcal proteins SP0010, SP0043, SP0079, SP0084,SP0092, SP0098, SP0106, SP0107, SP0127, SP0149, SP0191, SP0198, SP0249,SP0321, SP0346, SP0402, SP0453, SP0564, SP0582, SP0589, SP0601, SP0604,SP0617, SP0620, SP0629, SP0648, SP0659, SP0662, SP0664, SP0678, SP0724,SP0742, SP0757, SP0785, SP0787, SP0872, SP0878, SP0899, SP1002, SP1026,SP1032, SP1069, SP1154, SP1267, SP1376, SP1386, SP1404, SP1405, SP1419,SP1479, SP1500, SP1545, SP1560, SP1624, SP1652, SP1683, SP1826, SP1872,SP1891, SP1897, SP1942, SP1966, SP1967, SP1998, SP2048, SP2050, SP2083,SP2084, SP2088, SP2145, SP2151, SP2187, SP2192, SP2197, SP2207 andSP2218, or fragments thereof. In some embodiments, the pneumococcalantigen has an amino acid sequence selected from any or a combinationfrom SEQ ID NO: 1-76, or functional fragments thereof. In someembodiments, the pneumococcus antigen corresponding to SEQ ID NO: 1-76are encoded by nucleic acids of SEQ ID NO: 77-152. In some embodiments,fragments of the pneumococcal antigen are encompassed for use in themethods and compositions as disclosed herein, for example, pneumococcusantigens corresponding to SEQ ID NO: 153-234 as disclosed in Table 1.Other functional fragments of the pneumococcus antigens are encompassedfor use in the methods and compositions as disclosed herein, and can beassessed by one of ordinary skill in the art to determine if theyprovide protection against pneumococcus colonization, according to themethods as disclosed in Example 3 herein. A functional fragment of apneumococcal antigen of SEQ ID NO: 1-76 or SEQ ID NO: 153-234 can alsobe assessed by one of ordinary skill in the art for protection againstan invasive disease, such as sepsis according to the methods asdisclosed in Example 4 and 5, in particular when used alone or as partof a fusion protein with PdT. In some embodiments, a functional fragmentof a pneumococcal antigen of SEQ ID NO: 1-76 or SEQ ID NO: 153-234 canalso be assessed by one of ordinary skill in the art for protectionagainst an S. typhi according to the methods as disclosed in Example 5,in particular when used alone or as part of a fusion protein with PdTand/or fused to Vi.

In some embodiments, an immunogenic composition comprises at least oneisolated pneumococcal antigen selected from any one or a combination ofthe pneumococcal antigens with the amino acid sequences SEQ ID NO: 1-76or SEQ ID NO: 153-234. In some embodiments, the pneumococcal antigensare the full length pneumococcal proteins of SP0010, SP0043, SP0079,SP0084, SP0092, SP0098, SP0106, SP0107, SP0127, SP0149, SP0191, SP0198,SP0249, SP0321, SP0346, SP0402, SP0453, SP0564, SP0582, SP0589, SP0601,SP0604, SP0617, SP0620, SP0629, SP0648, SP0659, SP0662, SP0664, SP0678,SP0724, SP0742, SP0757, SP0785, SP0787, SP0872, SP0878, SP0899, SP1002,SP1026, SP1032, SP1069, SP1154, SP1267, SP1376, SP1386, SP1404, SP1405,SP1419, SP1479, SP1500, SP1545, SP1560, SP1624, SP1652, SP1683, SP1826,SP1872, SP1891, SP1897, SP1942, SP1966, SP1967, SP1998, SP2048, SP2050,SP2083, SP2084, SP2088, SP2145, SP2151, SP2187, SP2192, SP2197, SP2207and SP2218. In some embodiments, a pneumococcal antigen comprises apneumococcal protein that lacks a signal sequence and/or transmembranedomain. In some embodiments, a pneumococcal antigen comprises a mixtureof a full length pneumococcal proteins and fragments resulting fromprocessing, or partial processing of a signal sequence by an expressionhost, e.g., E. coli or an insect cell line (e.g., the baculovirusexpression system), or a mammalian (e.g., human or Chinese hamster Ovary(CHO)) cell line. As used herein, the terms “portion” and “fragment” orgrammatical equivalents are used interchangeably.

In some embodiments, the pneumococcal antigens are a fragment of thefull length pneumococcal proteins of SP0010, SP0043, SP0079, SP0084,SP0092, SP0098, SP0106, SP0107, SP0127, SP0149, SP0191, SP0198, SP0249,SP0321, SP0346, SP0402, SP0453, SP0564, SP0582, SP0589, SP0601, SP0604,SP0617, SP0620, SP0629, SP0648, SP0659, SP0662, SP0664, SP0678, SP0724,SP0742, SP0757, SP0785, SP0787, SP0872, SP0878, SP0899, SP1002, SP1026,SP1032, SP1069, SP1154, SP1267, SP1376, SP1386, SP1404, SP1405, SP1419,SP1479, SP1500, SP1545, SP1560, SP1624, SP1652, SP1683, SP1826, SP1872,SP1891, SP1897, SP1942, SP1966, SP1967, SP1998, SP2048, SP2050, SP2083,SP2084, SP2088, SP2145, SP2151, SP2187, SP2192, SP2197, SP2207 andSP2218, for example, at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100,150, 200, 250, 300, 350 or 400 consecutive amino acids of such proteins.In some embodiments, fragment of the full-length pneumococcal proteincorrespond to SEQ ID NO: 153-234. In some embodiments, a fragment of apneumococcal protein is a functional fragment of any of SEQ ID NO:153-234, for example, at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100,150, 200, 250, 300, 350 or 400 consecutive amino acids of pneumococcalantigens corresponding to SEQ ID NO: 153-234.

In some embodiments, a pneumococcal antigen comprises an amino acidsequence which is at least 60% (e.g., at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98% or at least 99%) identical to at 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 100, 150, 200, 250, 300, 350 or 400 consecutiveamino acids of the pneumococcal proteins of SP0010, SP0043, SP0079,SP0084, SP0092, SP0098, SP0106, SP0107, SP0127, SP0149, SP0191, SP0198,SP0249, SP0321, SP0346, SP0402, SP0453, SP0564, SP0582, SP0589, SP0601,SP0604, SP0617, SP0620, SP0629, SP0648, SP0659, SP0662, SP0664, SP0678,SP0724, SP0742, SP0757, SP0785, SP0787, SP0872, SP0878, SP0899, SP1002,SP1026, SP1032, SP1069, SP1154, SP1267, SP1376, SP1386, SP1404, SP1405,SP1419, SP1479, SP1500, SP1545, SP1560, SP1624, SP1652, SP1683, SP1826,SP1872, SP1891, SP1897, SP1942, SP1966, SP1967, SP1998, SP2048, SP2050,SP2083, SP2084, SP2088, SP2145, SP2151, SP2187, SP2192, SP2197, SP2207and SP2218, or fragments thereof, e.g., fragments of pneumococcalantigens corresponding to SEQ ID NO: 153-234.

The inventors demonstrate herein that the pneumococcal antigen SP0785showed IL-17 response to protein stimulation and protection againstpneumococcal colonization, and that SP0785-PdT protected 80% of micefrom sepsis infection and SP0785-PdT-Vi protected against Salmonellatyphi infection. The inventors also demonstrated that the pneumococcalantigen SP1500 elicited an IL-17 response to protein stimulation andprotected against pneumococcal colonization, and also resulted inprotection of 50% of mice from from pneumococcal infection, and that thepneumococcal antigen SP0346 protected 60% of mice from from pneumococcalinfection and sepsis, that the pneumococcal antigens SP1386, SP0084 andSP1479 protected 50% of mice from from pneumococcal infection. Theinventors also demonstrated that SP2145, when conjugated to PdT(SP2145-PdT) protected 80% of mice from sepsis infection and thatSP2145-PdT-Vi protected against S. typhi infection. Accordingly, theseantigens provide novel compositions for eliciting immune responses withthe aim of eliciting beneficial immune responses, e.g., to protectagainst pneumococcal infections and associated pathogens. These antigensprovide novel targets for characterizing pneumococcal infections andimmune responses to pneumococcal infections.

Accordingly, in one aspect of the invention provides an immunogeniccomposition (e.g., vaccine) comprising an isolated pneumococcal antigenselected from a SP0785 polypeptide antigen, a SP1500 polypeptideantigen, a SP0346 polypeptide antigen, a SP1386 polypeptide antigen, aSP0084 polypeptide antigen, a SP1479 polypeptide antigen, a SP2145polypeptide antigen, and combinations thereof.

In some embodiments, an immunogenic composition comprises a SP0785pneumococcal polypeptide antigen. In some embodiments, a SP0785polypeptide antigen comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95,100, 150, 200, 250, 300, 350, or 400 consecutive amino acids of a SP0785polypeptide sequence. In some embodiments, a SP0785 polypeptide antigencomprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250,300, 350, or 400 consecutive amino acids of the sequence shown in SEQ IDNO:34. In some embodiments, a SP0785 polypeptide antigen comprises anamino acid sequence that is at least 60% (e.g., at least 65%, 70%, 75%,80%, 85%, 90%, 95%, or 98%) identical to at least 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80,85, 90, 95, 100, 150, 200, 250, 300, 350, or 400 consecutive amino acidsof the sequence shown in SEQ ID NO:35. In some embodiments, a SP0785pneumococcal polypeptide antigen is encoded by SEQ ID NO: 110 or afragment thereof. In some embodiments, functional fragment of a SP0785pneumococcal polypeptide antigen comprises amino acids of SEQ ID NO: 190or comprises an amino acid sequence that is at least 60% (e.g., at least65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) identical to at least 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60,65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, or 400consecutive amino acids of the sequence shown in SEQ ID NO:190.

In some embodiments, an immunogenic composition comprises a SP1500pneumococcal polypeptide antigen. In some embodiments, a SP1500polypeptide antigen comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95,100, 150, 200, 250, 300, 350, or 400 consecutive amino acids of a SP1500polypeptide sequence. In some embodiments, a SP1500 polypeptide antigencomprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250,300, 350, or 400 consecutive amino acids of the sequence shown in SEQ IDNO:51. In some embodiments, a SP1500 polypeptide antigen comprises anamino acid sequence that is at least 60% (e.g., at least 65%, 70%, 75%,80%, 85%, 90%, 95%, or 98%) identical to at least 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80,85, 90, 95, 100, 150, 200, 250, 300, 350, or 400 consecutive amino acidsof the sequence shown in SEQ ID NO:51. In some embodiments, a SP1500pneumococcal polypeptide antigen is encoded by SEQ ID NO: 127 or afragment thereof. In some embodiments, functional fragment of a SP1500pneumococcal polypeptide antigen comprises amino acids of SEQ ID NO: 209or comprises an amino acid sequence that is at least 60% (e.g., at least65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) identical to at least 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60,65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, or 400consecutive amino acids of the sequence shown in SEQ ID NO:209.

In some embodiments, an immunogenic composition comprises a SP0346pneumococcual polypeptide antigen. In some embodiments, a SP0346polypeptide antigen comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95,100, 150, 200, 250, 300, 350, or 400 consecutive amino acids of a SP0346polypeptide sequence. In some embodiments, a SP0346 polypeptide antigencomprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250,300, 350, or 400 consecutive amino acids of the sequence shown in SEQ IDNO:15. In some embodiments, a SP0346 polypeptide antigen comprises anamino acid sequence that is at least 60% (e.g., at least 65%, 70%, 75%,80%, 85%, 90%, 95%, or 98%) identical to at least 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80,85, 90, 95, 100, 150, 200, 250, 300, 350, or 400 consecutive amino acidsof the sequence shown in SEQ ID NO:15. In some embodiments, a SP0346pneumococcal polypeptide antigen is encoded by SEQ ID NO: 91 or afragment thereof. In some embodiments, functional fragment of a SP0346pneumococcal polypeptide antigen comprises amino acids of SEQ ID NO: 166or comprises an amino acid sequence that is at least 60% (e.g., at least65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) identical to at least 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60,65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, or 400consecutive amino acids of the sequence shown in SEQ ID NO:166.

In some embodiments, an immunogenic composition comprises a SP1386pneumococcal polypeptide antigen. In some embodiments, a SP1386polypeptide antigen comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95,100, 150, 200, 250, 300, 350, or 400 consecutive amino acids of a SP1386polypeptide sequence. In some embodiments, a SP1386 polypeptide antigencomprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250,300, 350, or 400 consecutive amino acids of the sequence shown in SEQ IDNO:46. In some embodiments, a SP1386 polypeptide antigen comprises anamino acid sequence that is at least 60% (e.g., at least 65%, 70%, 75%,80%, 85%, 90%, 95%, or 98%) identical to at least 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80,85, 90, 95, 100, 150, 200, 250, 300, 350, or 400 consecutive amino acidsof the sequence shown in SEQ ID NO:46. In some embodiments, a SP1386pneumococcal polypeptide antigen is encoded by SEQ ID NO: 122 or afragment thereof. In some embodiments, functional fragment of a SP1386pneumococcal polypeptide antigen comprises amino acids of SEQ ID NO: 204or comprises an amino acid sequence that is at least 60% (e.g., at least65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) identical to at least 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60,65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, or 400consecutive amino acids of the sequence shown in SEQ ID NO: 204.

In some embodiments, an immunogenic composition comprises a SP0084pneumococcal polypeptide antigen. In some embodiments, a SP0084polypeptide antigen comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95,100, 150, 200, 250, 300, 350, or 400 consecutive amino acids of a SP0084polypeptide sequence. In some embodiments, a SP0084 polypeptide antigencomprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250,300, 350, or 400 consecutive amino acids of the sequence shown in SEQ IDNO:4. In some embodiments, a SP0084 polypeptide antigen comprises anamino acid sequence that is at least 60% (e.g., at least 65%, 70%, 75%,80%, 85%, 90%, 95%, or 98%) identical to at least 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80,85, 90, 95, 100, 150, 200, 250, 300, 350, or 400 consecutive amino acidsof the sequence shown in SEQ ID NO:4. In some embodiments, a SP0084pneumococcal polypeptide antigen is encoded by the sequence shown in SEQID NO: 80 or a fragment thereof. In some embodiments, functionalfragment of a SP0084 pneumococcal polypeptide antigen comprises aminoacids of SEQ ID NO: 156 or comprises an amino acid sequence that is atleast 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%)identical to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200,250, 300, 350, or 400 consecutive amino acids of the sequence shown inSEQ ID NO:156.

In some embodiments, an immunogenic composition comprises a SP1479pneumococcal polypeptide antigen. In some embodiments, a SP1479polypeptide antigen comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95,100, 150, 200, 250, 300, 350, or 400 consecutive amino acids of a SP1479polypeptide sequence. In some embodiments, a SP1479 polypeptide antigencomprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250,300, 350, or 400 consecutive amino acids of the sequence shown in SEQ IDNO:50. In some embodiments, a SP1479 polypeptide antigen comprises anamino acid sequence that is at least 60% (e.g., at least 65%, 70%, 75%,80%, 85%, 90%, 95%, or 98%) identical to at least 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80,85, 90, 95, 100, 150, 200, 250, 300, 350, or 400 consecutive amino acidsof the sequence shown in SEQ ID NO:50. In some embodiments, a SP1479pneumococcal polypeptide antigen is encoded by the sequence shown in SEQID NO: 126 or a fragment thereof. In some embodiments, functionalfragment of a SP1479 pneumococcal polypeptide antigen comprises aminoacids of SEQ ID NO: 208 or comprises an amino acid sequence that is atleast 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%)identical to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200,250, 300, 350, or 400 consecutive amino acids of the sequence shown inSEQ ID NO: 208.

In some embodiments, an immunogenic composition comprises a SP2145pneumococcal polypeptide antigen. In some embodiments, a SP2145polypeptide antigen comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95,100, 150, 200, 250, 300, 350, or 400 consecutive amino acids of a SP2145polypeptide sequence. In some embodiments, a SP2145 polypeptide antigencomprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250,300, 350, or 400 consecutive amino acids of the sequence shown in SEQ IDNO:70. In some embodiments, a SP2145 polypeptide antigen comprises anamino acid sequence that is at least 60% (e.g., at least 65%, 70%, 75%,80%, 85%, 90%, 95%, or 98%) identical to at least 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80,85, 90, 95, 100, 150, 200, 250, 300, 350, or 400 consecutive amino acidsof the sequence shown in SEQ ID NO:70. In some embodiments, a SP2145pneumococcal polypeptide antigen is encoded by the sequence shown in SEQID NO: 146 or a fragment thereof. In some embodiments, functionalfragment of a SP2145 pneumococcal polypeptide antigen comprises aminoacids of SEQ ID NO: 70 or comprises an amino acid sequence that is atleast 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%)identical to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200,250, 300, 350, or 400 consecutive amino acids of the sequence shown inSEQ ID NO: 70.

In some embodiments, an immunogenic composition comprises two or moreisolated pneumococcal antigens. In some embodiments, the two or moreisolated antigens comprise two or more of a polypeptide antigensselected from Table 1. In some embodiments, the two or more isolatedpneumococcal antigens comprise three or more of a polypeptide antigenselected from Table 1. In some embodiments, the two or more isolatedpneumococcal antigens comprise four or more of a polypeptide antigenselected from Table 1. In some embodiments, the two or more isolatedpneumococcal antigens comprise five, six, seven or more of a polypeptideantigen selected from Table 1. In some embodiments, the two or moreisolated pneumococcal antigens comprise eight polypeptide antigensselected from Table 1.

The inventive pneumococcal antigens as described herein may be used inconjunction with other pneumococcal antigens such as those known in theart, such as those disclosed in US patent WO/2000/037105, U.S. Pat. Nos.7,217,791 and 7,585,669, US2006/0121058, US2012/0251577, US2011/0159040,US2012/0189649, and US20110020386 which are incorporated herein in theirentirety by reference. Other appropriate S. pneumoniae antigens forcombination vaccines include Pneumococcal surface protein A (PspA);derivatives of PspA, Choline-binding protein A (CbpA) and derivativesthereof; Pneumococcal surface adhesin A (PsaA); caseinolytic protease;sortase A (SrtA); pilus 1 RrgA adhesin; PpmA; PrtA; PavA; LytA; Stk-PR;PcsB; RrgB and derivatives thereof. For further details, see, e.g., A. DOgunniyi et al., “Protection against Streptococcus pneumoniae elicitedby immunization with pneumolysin and CbpA,” Infect Immun. 2001 October;69 (10):5997-6003; which is incorporated by reference herein in itsentirety.

Derivatives of PspA include proline-rich segments with the non-prolineblock (PR+NPB, further described below as well as in Daniels, C. C. etal. (2010) Infection and Immunity 78:2163-72) and related constructscomprising all or a fragment of the proline-rich region of PspA (e.g.,regions containing one or more of the sequences PAPAP (SEQ ID NO: 262),PKP, PKEPEQ (SEQ ID NO: 402) and PEKP (SEQ ID NO: 403) and optionallyincluding a non-proline block). An example of the non-proline-block hasthe exemplary sequence EKSADQQAEEDYARRSEEEYNRLTQQQ (SEQ ID NO: 239),which generally has no proline residues in an otherwise proline-richarea of the non-coiled region of PspA. PspA and its derivatives caninclude genes expressing similar proline-rich structures (i.e. PKP,PKEPEQ (SEQ ID NO: 402) and PEKP (SEQ ID NO: 403)), with or without theNPB. The amino acids at either end of the NPB mark the boundaries of theproline-rich region. In one example, the amino-terminal boundary to thePR-region is DLKKAVNE (SEQ ID NO: 240), and the carboxy-terminalboundary is (K/G)TGW(K/G)QENGMW (SEQ ID NO: 241). Peptides containingthe NPB are particularly immunogenic, suggesting that the NPB may be animportant epitope. Exemplary immunogenic PspA polypeptides andderivatives thereof (e.g. with and without the coiled-coil structure aredescribed, e.g., in International Patent Publication WO2013109995; whichis incorporated by reference herein in its entirety Immunogenic PspApolypeptides include both PR and NPB sequences (PR+NPB) Immunogenic PspApolypeptides can include only a PR sequence (PR only) and lack the NPB.

In some embodiments, an immunogenic composition comprises an isolatedpneumococcal polypeptide antigen selected from Table 1. In someembodiments, an immunogenic composition comprises two, three, four, fiveor more isolated pneumococcal polypeptide antigens selected fromTable 1. In some embodiments, a pneumococcal antigen is fused to aheterologous polypeptide (e.g., an epitope tag). In some embodiments, animmunogenic composition comprising a pneumococcal antigen includes apharmaceutically acceptable excipient.

While pneumococcal antigens are disclosed in WO/2000/037105, unlike inthe present invention, in WO/2000/037105 their ability to induce a Th17cell response was not assessed or known. Additionally, pneumococcalantigens are also disclosed in US application 2012/0189649 andUS2012/0251577. However unlike the present invention, in the '649 and'577 applications, a whole genome library was used instead of purifiedproteins to screen for antigens which elicited a Th17 response, thushigh expression levels of the antigens are likely to be required for animmunogenic effect or to elicit a Th17 response. In the presentinvention, purified pneumococcal antigen proteins are used, and thus hasan advantage of very low background and requires lower expression levelsof each pneumococcal antigen to induce an immunogenic effect and elicita Th17 response, as demonstrated in the Examples.

The pneumococcal antigens as described herein can be prepared usingrecombinant DNA technology, purified from natural sources, orsynthesized chemically. In some embodiments, where a pneumococcalantigen as described herein is fused or conjugated to PdT, the PdT candiffer in amino acid sequence from a naturally occurring S. pneumoniaepneumolysin protein.

Nucleic acids encoding truncated and/or mutated forms of a pneumococcalantigen as described herein can be prepared, for example, by polymerasechain reaction (PCR). Nucleic acids encoding such proteins can be chosenfor having codons, which are preferred or non-preferred, for aparticular expression system. For example, the nucleic acid can be onein which at least one codon, preferably at least 10%, or 20% of thecodons have been altered such that the sequence is optimized forexpression in E. coli, yeast, human, insect, or CHO cells.

Nucleic acids encoding truncated and/or mutated forms of a pneumococcalantigen as described herein can be fused to nucleotide sequencesencoding (1) other pneumococcal proteins, such as autolysin, surfaceprotein A, neuraminidase, hyaluronate lysate, choline binding protein A,or (2) non-pneumococcal proteins from organisms such as hemophilusinfluenza b, meningococcus group A, B, or C, or streptococcus group B.The nucleic acids encoding such fused protein are expressed in theexpression systems.

Pneumolysin truncates can be useful carriers of polysaccharides, ashosts may lacking pre-existing antibodies to such a carrier polypeptide.Pneumolysin is a virulence factor in pneumococcal infections and thereis little antigenic variation of the pneumolysin among pneumococci withdifferent subtypes.

A pneumococcal antigen as described herein, when administered to amammal such as a human, when fused to a polysaccharide induces immuneresponse that exceeds in magnitude, type, and/or duration the immuneresponse induced by administration to a mammal of only thepolysaccharide component. Accordingly, the pneumococcal antigencomponent must be of a length sufficient to induce such an enhancedimmune response. For fragments of a naturally occurring pneumococcalantigen as described herein, the fragments are at least 8, 10, 25, 50,75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 425, 450, 460, 465,460, 465, or more amino acids in length. For pneumococcal antigens,varying in sequence from a naturally occurring S. pneumoniae antigens asdescribed herein, the polypeptide can be at least about 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more identical to a naturallyoccurring S. pneumococcal antigens as described herein in Table 1.

The polysaccharide component which can be conjugated to one or more of apneumococcal antigen as described herein can be any S. pneumoniaecapsular polysaccharide, including but not limited to, any of subtypes1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A,19F, 19A, 20, 22F, 23A, 23F, 24F, 27, 33F, or 34. In some embodiments,the capsular polysaccharide is selected from subtypes 4, 6B, 9V, 14,18C, 19F, or 23F. In some embodiments, the polysaccharide is serotype14. In other embodiments, the polysaccharide is serotype 18C. One ormore of different capsular polysaccharides can be conjugated to a singlepolypeptide or a plurality of polypeptides. For example, a multivalentconjugate can include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10different capsular polysaccharides. Polysaccharides can be conjugated topolypeptides, for example, via a monomeric linkage (only one end of thepolysaccharide is attached to the polypeptide), a looped linkage (asingle polypeptide is attached to looped polysaccharides), orcross-linked (multiple polysaccharides attached to multiplepolypeptides).

Methods for the purification of polypeptides, e.g., a pneumococcalantigen as described herein, can be performed by one of ordinary skillin the art, and polypeptide or polysaccharide purification andconjugation processes are described in, e.g., U.S. Pat. Nos. 4,242,501;4,686,102; 5,623,057; and 5,565,204, which are incorporated herein inits entirety by reference.

The immunogenic compositions described herein can be administered to amammal to elicit an immune response (a prophylactic and/or therapeuticimmune response) against S. pneumoniae in the mammal. A pharmaceuticalcomposition containing a pneumococcal antigen, alone or as a conjugatecan be delivered in a pharmaceutically acceptable carrier, buffer, orpreservative which is suitable for a vaccine including, but not limitedto, physiological saline or other injectable liquids. Additivescustomary in vaccines may also be present, for example stabilizers suchas lactose or sorbitol, and adjuvants to enhance the immunogenicresponse such as aluminum phosphate, hydroxide, or sulphate and stearyltyrosine. The vaccine produced may also be used as components ofmultivalent vaccines which elicit an immune response against a pluralityof infectious agents.

The compositions can be administered in any manner known in the art,e.g., orally intramuscularly, intravenously, intraarterially,intrathecally, intradermally, intraperitoneally, intranasally,intrapulmonarily, intraocularly, intravaginally, intrarectally orsubcutaneously. They can be introduced into the gastrointestinal tractor the respiratory tract, e.g., by inhalation of a solution or powdercontaining the conjugates. In some embodiments, the compositions can beadministered via a skin patch.

A pharmaceutical composition (e.g., a vaccine) is administered in anamount sufficient to elicit production of antibodies as part of animmunogenic response. Dosage for any given patient depends upon manyfactors, including the patient's size, general health, sex, body surfacearea, age, the particular compound to be administered, time and route ofadministration, and other drugs being administered concurrently.Determination of optimal dosage is well within the abilities of apharmacologist of ordinary skill.

The ability of a composition to elicit an immune response in a hostmammal can be assayed by using methods for measuring immune responsesthat are well known in the art. For example, the generation of cytotoxicT cells can be demonstrated in a standard ⁵¹Cr release assay, bymeasuring intracellular cytokine expression or secretion, or by usingmajor histocompatibility complex (MHC) tetramers. Standard assays, suchas enzyme-linked immunosorbent assay (ELISA) or enzyme-linked immunospot(ELISPOT), can be used to measure cytokine profiles attributable to Tcell activation. T cell proliferation can be measured using assays suchas ³H-thymidine uptake and other assays known in the art. B cellresponses can be measured using art recognized assays such as ELISA.Other methodologies can also be used to evaluate the effects of theconjugates on pathogen-associated lesions or on other pathogen levelsgenerally (e.g., pneumococci clearance in challenged mice treated withthe conjugate).

The composition described herein can be used in the manufacture of amedicament for the prevention or treatment of an infection with S.pneumoniae or conditions associated with such infection.

Adjuvants

In some embodiments, an immunogenic composition comprising apneumococcal antigen includes an adjuvant. In some embodiments, animmunogenic composition includes a mineral-containing adjuvant. In someembodiments, the mineral-containing adjuvant includes aluminumhydroxide. In some embodiments, an immunogenic composition includes anadjuvant comprising an immunomodulatory oligonucleotide. In someembodiments, an immunogenic composition includes IC31™ adjuvant(Intercell AG). In some embodiments, an immunogenic composition includesan adjuvant comprising a toxin. In some embodiments, an immunogeniccomposition includes an adjuvant comprising an endotoxin. In someembodiments, an immunogenic composition includes an adjuvant comprisinga muramyl dipeptide. In some embodiments, an immunogenic compositionincludes an adjuvant comprising an oil emulsion. In some embodiments, animmunogenic composition includes an adjuvant comprising a saponin. Insome embodiments, an immunogenic composition includes an adjuvantcomprising an immune stimulating complex (ISCOM). In some embodiments,an immunogenic composition includes an adjuvant comprising a nonionicblock copolymer. In some embodiments, an immunogenic compositionincludes virus-like particles (VLPs), for example, a cholera toxin. Insome embodiments, an immunogenic composition includes replicons. In someembodiments, an immunogenic composition includes an adjuvant comprisingliposomes. In some embodiments, an immunogenic composition includes anadjuvant comprising microparticles. In some embodiments, an immunogeniccomposition includes an adjuvant comprising biodegradable microspheres.In some embodiments, an immunogenic composition includes an adjuvantcomprising a cytokine. In some embodiments, an immunogenic compositionincludes an adjuvant comprising a lipopeptide.

Immunogenic compositions may contain adjuvants. In some embodiments,cholera toxin (CT) can be used as an adjuvant for intranasaladministration, resulting in protection from pneumococcal colonization.Alum is an affective adjuvant for subcutaneous injection. Adjuvants aretypically a heterogeneous group of substances that enhance theimmunological response against an antigen that is administeredsimultaneously. In some instances, adjuvants are added to a vaccine toimprove the immune response so that less vaccine is needed. Adjuvantsserve to bring the antigen—the substance that stimulates the specificprotective immune response—into contact with the immune system andinfluence the type of immunity produced, as well as the quality of theimmune response (magnitude or duration). Adjuvants can also decrease thetoxicity of certain antigens and provide solubility to some vaccinecomponents. Almost all adjuvants used today for enhancement of theimmune response against antigens are particles or form particlestogether with the antigen. In the book “Vaccine Design—the subunit andadjuvant approach” (Ed: Powell & Newman, Plenum Press, 1995) almost allknown adjuvants are described both regarding their immunologicalactivity and regarding their chemical characteristics. The type ofadjuvants that do not form particles are a group of substances that actas immunological signal substances and that under normal conditionsconsist of the substances that are formed by the immune system as aconsequence of the immunological activation after administration ofparticulate adjuvant systems.

Adjuvants for vaccines are well known in the art. Suitable additionaladjuvants include, but are not limited to: complete Freund's adjuvant(CFA), incomplete Freund's adjuvant (IFA), saponin, mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyaninons, peptides, oil or hydrocarbon emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum. Selection of an adjuvant depends on the animal subject to bevaccinated. Additional examples include, but are not limited to,monoglycerides and fatty acids (e. g. a mixture of mono-olein, oleicacid, and soybean oil); mineral salts, e.g., aluminium hydroxide andaluminium or calcium phosphate gels; oil emulsions and surfactant basedformulations, e.g., MF59 (microfluidised detergent stabilizedoil-in-water emulsion), QS21 (purified saponin), AS02 [SBAS2](oil-in-water emulsion+MPL+QS-21), Montanide ISA-51 and ISA-720(stabilized water-in-oil emulsion); particulate adjuvants, e.g.,virosomes (unilamellar liposomal vehicles incorporating influenzahaemagglutinin), AS04 ([SBAS4] Al salt with MPL), ISCOMS (structuredcomplex of saponins and lipids), polylactide co-glycolide (PLG);microbial derivatives (natural and synthetic), e.g., monophosphoryllipid A (MPL), Detox (MPL+M. Phlei cell wall skeleton), AGP [RC-529](synthetic acylated monosaccharide), DC_Chol (lipoidal immunostimulatorsable to self organize into liposomes), OM-174 (lipid A derivative), CpGmotifs (synthetic oligonucleotides containing immunostimulatory CpGmotifs), modified LT and CT (genetically modified bacterial toxins toprovide non-toxic adjuvant effects); endogenous human immunomodulators,e.g., hGM-CSF or hIL-12 (cytokines that can be administered either asprotein or plasmid encoded), Immudaptin (C3d tandem array) and inertvehicles, such as gold particles. Newer adjuvants are described in U.S.Pat. No. 6,890,540, United States Patent Application No. 2005/0244420,and PCT/SE97/01003, and US2012/0135025, the contents of which areincorporated herein in their entirety by reference. The adjuvant canalso be selected from the group consisting of QS-21, Detox-PC, MPL-SE,MoGM-CSF, TiterMax-G, CRL-1005, GERBU, TERamide, PSC97B, Adjumer,PG-026, GSK-I, GcMAF, B-alethine, MPC-026, Adjuvax, CpG ODN, Betafectin,Alum, and MF59.

In some embodiments, alternative adjuvants can be used, such as apharmaceutically acceptable adjuvant. For example, oils or hydrocarbonemulsion adjuvants should not be used for human vaccination. One exampleof an adjuvant suitable for use with humans is alum (alumina gel).Details of common adjuvants which are contemplated to be added to thevaccine comprising an antigen as disclosed in Table 1 include thosediscussed below:

Complete Freund's Adjuvant (CFA): A mineral oil adjuvant; uses awater-in-oil emulsion which is primarily oil. For many years theadjuvant of choice was complete Freund's adjuvant. This adjuvant, whilepotent immunogenically, also has had a significant history of frequentlyproducing abscesses, granulomas and tissue sloughs. It contains paraffinoil, killed mycobacteria and mannide monoosleate. The paraffin oil isnot metabolized; it is either expressed through the skin (via agranuloma or abscess) or phagocytized by macrophages. Multiple exposuresto CFA will cause severe hypersensitivity reactions. Accidental exposureof personnel to CFA can result in sensitization to tuberculin.

Incomplete Freund's Adjuvant (IFA): Also a mineral oil adjuvant.Composition similar to CFA but does not contain the killed mycobacteriaso does not produce as severe reactions. Used for the boosterimmunizations following the initial injection with antigen-CFA. IFA canbe used for initial injection if the antigen is strongly immunogenic.

Montanide ISA (Incomplete Seppic Adjuvant): A mineral oil adjuvant. Usesmannide oleate as the major surfactant component. The antibody responseis generally similar to that with IFA. Montanide ISA may have a lessenedinflammatory response.

Ribi Adjuvant System (RAS): An oil-in-water emulsion that containsdetoxified endotoxin and mycobacterial cell wall components in 2%squalene. Multiple formulations are commercially available, dependent onuse. Is an alternative to CFA. Lower viscosity than CFA. Results(titers) often comparable to those with CFA. The squalene oil ismetabolizable. RAS has a lower incidence of toxic reactions.

TiterMax: Another water-in-oil emulsion, this preparation combines asynthetic adjuvant and microparticulate silica with the metabolizableoil squalene. The copolymer is the immunomodulator component. Antigen isbound to the copolymer and presented to the immune cells in a highlyconcentrated form. Less toxicity than CFA. TiterMax usually produces thesame results as CFA.

Syntex Adjuvant Formulation (SAF): A preformed oil-in-water emulsion.Uses a block copolymer for a surfactant. A muramyl dipeptide derivativeis the immunostimulatory component. All in squalene, a metabolizableoil. SAF can bias the humoral response to IgG2a in the mouse, but isless toxic than CFA.

Aluminum Salt Adjuvants: Most frequently used as adjuvants for vaccineantigen delivery. Generally weaker adjuvants than emulsion adjuvants.Aluminum Salt Adjuvants are best used with strongly immunogenicantigens, but result generally in mild inflammatory reactions.

Nitrocellulose-adsorbed antigen: The nitrocellulose is basically inert,leading to almost no inflammatory response. Slow degradation ofnitrocellulose paper allows prolonged release of antigen. Does notproduce as dramatic an antibody response as CFA. Nitrocellulose-adsorbedantigen is good for use if only a small amount of antigen can berecovered from a gel band, e.g., for animal immunization.

Encapsulated or entrapped antigens: Permits prolonged release of antigenover time; can also have immunostimulators in preparation for prolongedrelease. Preparation of encapsulated or entrapped antigens is complex.

Immune-stimulating complexes (ISCOMs): Antigen modifiedsaponin/cholesterol micelles. Stable structures are formed which rapidlymigrate to draining lymph nodes. Both cell-mediated and humoral immuneresponses are achieved. Low toxicity; ISCOMs can elicit significantantibody response. Quil A is one example, QS-21 is another.

GerbuR adjuvant: An aqueous phase adjuvant which uses immunostimulatorsin combination with zinc proline. GerbuR does not have a depot effectand has minimal inflammatory effect. GerbuR requires frequent boostingto maintain high titers.

Another group of adjuvants include immune stimulators such as cytokinesIL-12, IL-4 and costimulatory molecules such as B7. A wide range ofmolecules having immune stimulating effects are known includingaccessory molecules such as ICAM and LFA. In some embodiments, GM-CSF isadministered to the patient before the initial immune administration.GM-CSF can be administered using a viral vector or an isolated proteinin a pharmaceutical formulation. Combinations of adjuvants can be usedsuch as CM-CSF, ICAM and LFA. While a strong immune response istypically generated to infectious disease antigens, tumor associatedantigens typically generate a weaker immune response. Thus, immunestimulators such as described above are preferably used with them.

In some embodiments, a pneumococcal vaccine can contain severalpneumococcal antigens and/or be formulated with different or noveladjuvants, or incorporated in vaccine scaffolds, such as afusion-conjugate (e.g., a fusion with a pneumolysoid and conjugation toa polysaccharide as proposed in Lu et al, Infection and Immunity, 2009),or using a scaffold, as disclosed in PCT/US12/37412 and WO 2012/155007(which are incorporated herein in their entirety by reference) toimprove immunogenicity and facilitate different routes ofadministration. Accordingly, a composition comprising at least 2, or atleast about 3, or at least about 4, or at least about 5, or at leastabout 6, or at least about 7, or at least about 8, or at least about 9,or at least about 10, or at least about 12, or at least about 14, or atleast about 16, or at least about 18, or at least about 20, or at leastabout 25, or any integer between about 2 and about 26 different antigensor more that 25 different antigens can be used, alone or in combinationwith an adjuvant and/or vaccine scaffold, such as a polysaccharide canbe used.

In another aspect, the invention provides methods for eliciting animmune response against pneumococcus in a mammal. The methods include,for example, administering to the mammal an immunogenic compositioncomprising an isolated pneumococcal polypeptide antigen selected fromTable 1 or fragments and combinations thereof, e.g., an immunogeniccomposition described herein.

In some embodiments, a method elicits an immune response againstpneumococcus. In some embodiments, a method elicits a T cell response toa pneumococcal antigen (e.g., a CD4+ T cell mediated immune responseand/or a CD8+ T cell mediated immune response). In some embodiments, amethod elicits a Th1 T cell response. In some embodiments, a methodelicits a Th17 T cell response. In some embodiments, a method elicitsIFN-γ secretion by antigen-specific T cells. In some embodiments, amethod elicits an antibody response (e.g., an IgG response, and/or anIgA response). In some embodiments, a method elicits a cytotoxic T cell(CTL) response. In some embodiments, a method elicits a B cell-mediatedimmune response. In some embodiments, a method elicits both a T cell-and a B cell-mediated response. In some embodiments, a method elicits aninnate immune response.

In some embodiments, a method reduces the incidence of pneumococcalinfection in subjects administered the composition. In some embodiments,a method reduces the likelihood of lower tract infection by apneumococcus. In some embodiments, a method reduces the likelihood ofupper tract infection by a pneumococcal organism. In some embodiments, amethod reduces the likelihood of chronic infection by a pneumococcalorganism. In some embodiments, a method reduces the likelihood ofsuffering from pelvic inflammatory disease due to a pneumococcalinfection. In some embodiments, a method reduces the likelihood ofinfertility subsequent to a pneumococcal infection.

In some embodiments of a method, an immunogenic composition isadministered to the mammal at least two times (e.g., two, three, four,or five times).

In some embodiments, an immunogenic composition administered after afirst administration (i.e., as a boost) differs from the compositionadministered initially, e.g., the composition includes a differentpneumococcal antigen or a different subset of pneumococcal antigens, ora different pneumococcal antigen substance (polypeptide or nucleic acidencoding same), or a different dose of antigen, or a different adjuvant,or a different dose of adjuvant. In some embodiments, a boost isadministered by a different route than a previous administration.

In some embodiments, the mammal is at risk for infection withpneumococcus. In some embodiments, the mammal is infected with apneumococcal infection. In some embodiments, the mammal is a human.

In some embodiments, an immunogenic composition administered in a methodcomprises an adjuvant. In some embodiments, an adjuvant is amineral-containing adjuvant. In some embodiments, an immunogeniccomposition administered in a method comprises a pharmaceuticallyacceptable excipient.

In some embodiments, a pneumococcal antigen composition includes anadjuvant. In some embodiments, the adjuvant includes mineral-containingadjuvant. Mineral-containing adjuvants can be formulated as gels, incrystalline form, in amorphous form, as particles, etc.Mineral-containing adjuvants include, for example, aluminum salts and/orcalcium salts (e.g., aluminum hydroxide, aluminum phosphate, aluminumsulfate, calcium phosphate, etc.). In some embodiments, a pneumococcalantigen composition includes aluminum hydroxide. Alhydrogel™ is anexample of an aluminum hydroxide gel adjuvant.

In some embodiments, an adjuvant includes an immunomodulatoryoligonucleotide. In some embodiments, an immunomodulatoryoligonucleotide sequence includes CpG (unmethylated cytosine-guanosine)motifs. Oligonucleotides having CpG motifs can include nucleotideanalogs and/or non-naturally occurring internucleoside linkages (e.g.,phosphorothioate linkages). For examples of various oligonucleotidesinclude CpG motifs, see Kandimalla, et al., Nuc. Acids Res. 31(9):2393-2400, 2003; WO02/26757; WO99/62923; Krieg, Nat. Med. 9(7): 831-835,2003; McCluskie, et al., FEMS Immunol Med. Microbiol. 32:179-185, 2002;WO98/40100; U.S. Pat. Nos. 6,207,646; 6,239,116 and 6,429,199. Otherimmunomodulatory nucleotide sequences double stranded RNA sequences,palindromic sequences, and poly(dG) sequences.

In some embodiments, an adjuvant comprises IC.sub.31™ (Intercell AG).IC31™ is a synthetic adjuvant that includes an antimicrobial peptide,KLK, and an immunostimulatory oligonucleotide, ODN1a, and acts as aToll-like Receptor 9 (TLR9) agonist. In some embodiments, an adjuvantincludes a toxin. In some embodiments, a toxin is a bacterialADP-ribosylating toxin, e.g., cholera toxin (CT), E. coli heat labiletoxin, or pertussis toxin. In some embodiments, the bacterial toxin is adetoxified form of an ADP-ribosylating toxin (see, e.g., Beignon, etal., Inf. Immun. 70(6):3012-3019, 2002; Pizza, et al., Vaccine19:2534-2541, 2001; Pizza, et al., Int. J. Med. Microbiol.290(4-5):455-461, 2000; Scharton-Kersten et al., Inf. Immun.68(9):5306-5313, 2000; Ryan et al., Inf. Immun 67(12):6270-6280, 1999;Partidos et al., Immunol Lett. 67(3):209-216, 1999; Peppoloni et al.,Vaccines 2(2):285-293, 2003; and Pine et al., J. Control Release85(1-3):263-270, 2002). In some embodiments, an adjuvant includes anendotoxin such as monophosphoryl lipid A or 3-De-O-acylatedmonophosphoryl lipid A (see U.S. Pat. No. 4,987,237 and GB 2122204B).

In some embodiments, an adjuvant includes a muramyl dipeptide (e.g.,N-acetyl-muramyl-L-threonyl-D-isoglutamine(thr-MDP),N-acetyl-normuramyl-1-alanyl-d-isoglutamine(nor-MDP), andN-acetylmuramyl-1-alanyl-d-isoglutaminyl-1-alanine-2-(1′-2′-dipalmitoyl-s-n-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE). In some, an adjuvant includes an oil emulsion and/oremulsifier-based adjuvant. In some embodiments, an oil emulsion adjuvantincludes a Freund's Adjuvant (e.g., Complete Freund's adjuvant (CFA), orincomplete Freund's adjuvant (IFA)). In some embodiments, anoil-emulsion adjuvant includes a squalene water emulsion, such as MF59(Novartis; see, e.g., WO9014837), or a Synex adjuvant formulation(SAF)). In some embodiments, an oil emulsion includes a dispersingagent, e.g., a mono- or di-C.sub.12-C.sub.24-fatty acid ester ofsorbitan or mannide, e.g., sorbitan mono-stearate, sorbitan mon-oleate,or mannide mono-oleate. Examples of oil emulsions that include squaleneand dispersing agents includes Arlacel™, Montanide™ ISA-720, andMontanide™ ISA-703. Other oil emulsions are described, e.g., in WO95/17210 and EP 0399842.

In some embodiments, an adjuvant includes a saponin. Saponins aresteroid and/or triterpenoid glycosides derived from plants such asQuillaja saponaria, Saponaria officianalis, Smilax ornata, andGypsophilla paniculata. Fractions of saponin-containing extracts thathave been described and that can be used as adjuvants for pneumococcalantigens include Quil™A, QS21, QS7, QS17, QS18, QH-A, QH-B, QH-C, andQuilA (see, e.g., U.S. Pat. No. 5,057,540). In some embodiments, QS21 isused as an adjuvant.

In some embodiments, an adjuvant includes an immune stimulating complex(ISCOM). ISCOMs are particles that typically include a glycoside (e.g.,a saponin) and a lipid. In some embodiments, an ISCOM includes a saponinand a cholesterol. In some embodiments, an ISCOM includes a saponin, acholesterol, and a phospholipid (e.g., phosphatidylcholine and/orphosphatidylethanolamine). In some embodiments, an ISCOM includes anonionic block copolymer. ISCOMs can include additional adjuvants, e.g.,additional adjuvant substances described herein (see, e.g., WO05/002620). In some embodiments, an ISCOM includes a substance thattargets it to a mucosal membrane (see, e.g., WO97/030728). Other ISCOMcompositions and preparation of the compositions suitable forcombination with pneumococcal antigens provided herein are described,e.g., in U.S. Pat. Pub. No. 20060121065, WO 00/07621, WO 04/004762, WO02/26255, and WO 06/078213. In some embodiments, an adjuvant comprisesan AbISCO® adjuvant (e.g., Matrix-M™, Isconova). In some embodiments, anadjuvant comprises AbISCO®-100. In some embodiments, an adjuvantcomprises AbISCO®-300.

In some embodiments, an adjuvant includes a nonionic block copolymer.Nonionic block copolymers typically include two chains of hydrophobicpolyoxyethylenes of various lengths combined with a block of hydrophobicpolyoxypropylene. In some embodiments, a nonionic block copolymer isformulated in an oil-in-water emulsion (e.g., with oil and squalene).

In some embodiments, an adjuvant includes virus like particles (VLPs).VLPs are non replicating, non infectious particles that typicallyinclude one or more viral proteins, optionally formulated with anadditional component such as a phospholipid. In some embodiments, a VLPincludes proteins from one or more of the following: an influenza virus(e.g., a hemaglutinin (HA) or neuraminidase (NA) polyptide), Hepatitis Bvirus (e.g., a core or capsid polypeptide), Hepatitis E virus, measlesvirus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus,Retrovirus, Norwalk virus, human papilloma virus, HIV, RNA-phages,Q13-phage (e.g., a coat protein), GA-phage, fr-phage, AP205 phage, a Ty(e.g., retrotransposon Ty protein p1). See, e.g., WO03/024480,WO03/024481, WO08/061,243, and WO07/098,186. In some embodiments, anadjuvant includes replicons. Replicons resemble VLPs in that they arenoninfectious particles including viral proteins, and further include anucleic acid encoding a polypeptide (e.g., an antigen). In someembodiments, a replicon includes proteins from an alphavirus.Alphaviruses include, e.g., Eastern Equine Encephalitis Virus (EEE),Venezuelan Equine Encephalitis Virus (VEE), Everglades Virus, MucamboVirus, Pixuna Virus, Western Equine Encephalitis Virus (WEE), SindbisVirus, Semliki Forest Virus, Middleburg Virus, Chikungunya Virus,O'nyong-nyong Virus, Ross River Virus, Barmah Forest Virus, Getah Virus,Sagiyama Virus, Bebaru Virus, Mayaro Virus, Una Virus, Aura Virus,Whataroa Virus, Babanki Virus, Kyzylagach Virus, Highlands J Virus, FortMorgan Virus, Ndumu Virus, and Buggy Creek Virus. In some embodiments,an adjuvant includes a replicon that includes a nucleic acid encodingone or more pneumococcal antigens described herein. In some embodiments,an adjuvant includes a replicon that encodes a cytokine (e.g.,interleukin-12 (IL-12), IL-23, or granulocyte-macrophagecolony-stimulating factor (GM-CSF)). Production and uses of repliconsare described, e.g., in WO08/058,035, WO08/085,557, and WO08/033,966).In some embodiments, a VLP or replicon adjuvant includes one or morepneumococcal antigens (i.e., VLP or replicon particles include apneumococcal antigen as part of the particles). In some embodiments, aVLP or replicon adjuvant is co-administered with a pneumococcal antigenpolypeptide.

In some embodiments, an adjuvant includes liposomes, which are areartificially-constructed spherical lipid vesicles (see, e.g., U.S. Pat.Nos. 4,053,585; 6,090,406; and 5,916,588). In certain embodiments, alipid to be used in liposomes can be, but is not limited to, one or aplurality of the following: phosphatidylcholine, lipid A, cholesterol,dolichol, sphingosine, sphingomyelin, ceramide, glycosylceramide,cerebroside, sulfatide, phytosphingosine, phosphatidyl-ethanolamine,phosphatidylglycerol, phosphatidylinositol, phosphatidylserine,cardiolipin, phosphatidic acid, and lyso-phosphatides. In someembodiments, an adjuvant includes a liposome and a ligand for aToll-like Receptor (TLR; see, e.g., WO/2005/013891, WO/2005/079511,WO/2005/079506, and WO/2005/013891). In some embodiments, an adjuvantincludes JVRS-100. JVRS-100 comprises cationic liposomes combined withnon-coding oligonucleotides or plasmids.

In some embodiments, an adjuvant includes microparticles comprised of apolymer, e.g., a polymer of acrylic or methacrylic acid,polyphosphazenes, polycarbonates, polylactic acid, polyglycolic acid,copolymers of lactic acid or glycolic acid, polyhydroxybutyric acid,polyorthoesters, polyanhydrides, polysiloxanes, polycaprolactone, or acopolymer prepared from the monomers of these polymers. In someembodiments, an adjuvant includes microparticles comprised of a polymerselected from the group consisting of polyvinylpyrrolidone,polyvinylalcohol, polyhydroxyethylmethacrylate, polyacrylamide,polymethacrylamide, and polyethyleneglycol (see, e.g., U.S. Pat. No.5,500,161).

In some embodiments, an adjuvant includes biodegradable microspheres(e.g., microspheres comprised of poly(D,L-lactic acid),poly(D,L-glycolic acid), poly(.epsilon.-caprolactone), polye(.alpha.-hydroxy actid), polyhydroxybutyric acid, a polyorthoester, apolyanhydride, etc.).

In some embodiments, an adjuvant includes a cytokine. In someembodiments, an adjuvant includes IL-12. In some embodiments, anadjuvant includes IL-23. In some embodiments, an adjuvant includesGM-CSF. In some embodiments, an adjuvant includes a lipopeptide. In someembodiments, an adjuvant includes a Pam-3-Cys lipopeptide. In someembodiments, an adjuvant including a lipopeptide activates Toll-likereceptors (TLRs).

Additional Components of Vaccines or Immunogenic Compositions

In addition to the antigens and the adjuvants described above, a vaccineformulation or immunogenic composition may include one or moreadditional components.

In certain embodiments, the vaccine formulation or immunogeniccomposition may include one or more stabilizers such as sugars (such assucrose, glucose, or fructose), phosphate (such as sodium phosphatedibasic, potassium phosphate monobasic, dibasic potassium phosphate, ormonosodium phosphate), glutamate (such as monosodium L-glutamate),gelatin (such as processed gelatin, hydrolyzed gelatin, or porcinegelatin), amino acids (such as arginine, asparagine, histidine,L-histidine, alanine, valine, leucine, isoleucine, serine, threonine,lysine, phenylalanine, tyrosine, and the alkyl esters thereof), inosine,or sodium borate.

In certain embodiments, the vaccine formulation or immunogeniccomposition includes one or more buffers such as a mixture of sodiumbicarbonate and ascorbic acid. In some embodiments, the vaccineformulation may be administered in saline, such as phosphate bufferedsaline (PBS), or distilled water.

In certain embodiments, the vaccine formulation or immunogeniccomposition includes one or more surfactants such as polysorbate 80(Tween 80), Triton X-100, Polyethylene glycol tert-octylphenyl ethert-Octylphenoxypolyethoxyethanol4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol (TRITON X-100);Polyoxyethylenesorbitan monolaurate Polyethylene glycol sorbitanmonolaurate (TWEEN 20); and 4-(1,1,3,3-Tetramethylbutyl)phenol polymerwith formaldehyde and oxirane (TYLOXAPOL). A surfactant can be ionic ornonionic.

In certain embodiments, the vaccine formulation or immunogeniccomposition includes one or more salts such as sodium chloride, ammoniumchloride, calcium chloride, or potassium chloride.

In certain embodiments, a preservative is included in the vaccine orimmunogenic composition. In other embodiments, no preservative is used.A preservative is most often used in multi-dose vaccine vials, and isless often needed in single-dose vaccine vials. In certain embodiments,the preservative is 2-phenoxyethanol, methyl and propyl parabens, benzylalcohol, and/or sorbic acid.

In certain embodiments, the vaccine formulation or immunogeniccomposition is a controlled release formulation.

Modifications

In some embodiments, the vaccine can comprise at least one immunogen ofthe sequences listed in Table 1, or an immunogen which is a functionalfragment or has substantial identity to an immunogen listed in Table 1.Further S. pneumoniae antigens as disclosed herein for combinationvaccines include conjugated S. pneumoniae polysaccharides. Theconjugated polysaccharides may be, for example, as described in U.S.Pat. Nos. 5,623,057, 5,371,197, or PCT/US2011/023526, which areincorporated herein in their entirety.

In some embodiments, the pneumococcal antigens described herein may beused with or without modification. In some embodiments, a pneumococcalantigen may be modified to elicit the desired immune response. In someembodiments, a pneumococcal antigen is conjugated to an appropriateimmunogenic carrier such as tetanus toxin, pneumolysin, keyhole limpethemocyanin, or the like.

In some embodiments, one or more pneumococcal antigen is present in ascaffold, such as a fusion-conjugate (e.g., a fusion with a pneumolysoidand conjugation to a polysaccharide as proposed in Lu et al, Infectionand Immunity, 2009), or using a scaffold, as disclosed inPCT/US12/37412, WO 2012/155007 and US2011/0206716, which areincorporated herein in its entirety by reference.

In some embodiments, a pneumococcal polypeptide antigen is fused to PdT.In some embodiments, PdT represents a nonhemolytic variant ofpneumolysin, or a nonhemolytic variant of pneumolysin (PdT) (W433F,D385N, and C428G).

In some embodiments, a pneumococcal polypeptide antigen can exist as aconjugate to provide for a synergistic immunogenic reaction to anantigenic sugar moiety. For example, the Vi polysaccharide of Salmonellatyphi could be used. Vi capsular polysaccharide has been developedagainst bacterial enteric infections, such as typhoid fever (Robbins etal., 150(3) J. Infect. Dis. 436-49 (1984); Levine et al., 7 BaillieresClin. Gastroenterol. 501-17 (1993)). Vi is a polymer ofα-1→4-galacturonic acid with an N acetyl at position C-2 and variable0-acetylation at C-3. The virulence of S. typhi correlates with theexpression of this molecule (Sharma et al., 101 P.N.A.S. USA 17492-97(2004)). The Vi polysaccharide vaccine of S. typhi has severaladvantages: Side effects are infrequent and mild, a single dose yieldsconsistent immunogenicity and efficacy. Vi polysaccharide may bereliably standardized by physicochemical methods verified for otherpolysaccharide vaccines, Vi is stable at room temperature and it may beadministered simultaneously with other vaccines without affectingimmunogenicity and tolerability (Azze et al., 21 Vaccine 2758-60(2003)).

Thus, the Vi polysaccharide of Salmonella typhi may be conjugated to afusion protein of a pneumococcal polypeptide antigen, where thepneumococcal polypeptide antigen is fused to PdT or another protein,such that the resulting vaccine confers immunity against one pathogen,or two different pathogens: where the pneumococcal polypeptide antigenconfers protection against pneumococcus, a Vi-pneumococcal antigen:PdTconstruct raises an immunogenic response against S. typhi andpneumococcus. Other examples include combining sugars from encapsulatedbacteria (such as meningococcus, S. aureus, pneumococcus, etc.) andtuberculous protein, to provide a vaccine that protects against twodifferent pathogens.

Other polysaccharide (PS) moities that may be used in the presentinvention in alternative to dextran, pneumococcal cell wallpolysaccharide (CWPS) etc., include carbohydrate antigens of cancers.For example, the Tn antigen, an oligosaccharide expressed exclusively bycancer cells (Buskus et al., 44 Angew Chem. Int'l Ed. 5985-88 (2005)).In some embodiment, the present invention provides for an immunogeniccomposition comprising a fusion protein of a truncated pneumococcalantigen and a nonhemolytic pneumolysin PdT protein, conjugated withCWPS.

In one aspect of the invention, the polysaccharide has a molecular massof <500 kDa. In another aspect of the invention, the PS has a molecularmass of <70 kDa.

In some embodiments, a pneumococcal antigen as disclosed herein ispresent in an immunogenic multiple antigen presenting system (MAPS) asdisclosed in WO 2012/155007, which is incorporated herein in itsentirety by reference, where the pneumococcal antigen as disclosedherein in present in an immunogenic complex comprising at least one typeof polymer, e.g., a polysaccharide, that can, optionally, be antigenic;at least one pneumococcal antigen as disclosed herein; and at least onecomplementary affinity-molecule pair comprising (i) a first affinitymolecule that associates with the polymer, and (ii) a complementaryaffinity molecule that associates with the protein or peptide; such thatthe first and complementary affinity molecules serve as an indirect linkbetween the polymer with the antigenic protein or peptide. Accordingly,the polymer can attach at least 1, or at least 2, or a plurality of thesame or different protein or peptide antigens. In some embodiments, thepolymer is antigenic, e.g., the polymer is a pneumococcal capsularpolysaccharide. In some embodiments, the pneumococcal antigens arerecombinant pneumococcal antigens. Accordingly, one aspect of thepresent invention relates to an immunogenic composition comprising apolymer, at least one pneumococcal antigen, and at least onecomplementary affinity-molecule pair, where the complementaryaffinity-molecule pair comprises a first affinity molecule thatassociates with the polymer and a complementary affinity molecule thatassociates with the pneumococcal antigen, so that when the firstaffinity molecule associates with the complementary affinity molecule,it indirectly links the antigen to the polymer.

In some embodiments, a first affinity molecule in a immunogenic complexis cross-linked to the polymer with a cross-linking reagent, forexample, a cross-linking reagent selected from CDAP(1-cyano-4-dimethylaminopyridinium tetrafluoroborate), EDC(1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride), sodiumcyanoborohydride; cyanogen bromide; or ammonium bicarbonate/iodoaceticacid. In some embodiments, the first affinity molecule is cross-linkedto carboxyl, hydroxyl, amino, phenoxyl, hemiacetal, and mecaptofunctional groups of the polymer. In some embodiments, the firstaffinity molecule is covalently bonded to the polymer. In someembodiments, the first affinity molecule is biotin or a derivativethereof, or a molecule with similar structure or physical property asbiotin, for example, an amine-PEG3-biotin((+)-biotinylation-3-6,9-trixaundecanediamine) or derivative thereof.

In some embodiments, a pneumococcal antigen of the immunogenic complexis a fusion protein comprising a pneumococcal antigenic protein orpeptide fused to the complementary affinity-binding molecule. The fusioncan be a genetic construct, i.e., a recombinant fusion peptide orprotein. In some embodiments, a pneumococcal antigen can be covalentlyattached as a fusion protein to the complementary affinity molecule. Inalternative embodiments, the antigen is non-covalently attached to thecomplementary affinity molecule.

In some embodiments, a complementary affinity molecule in an immunogeniccomplex is a biotin-binding protein or a derivative or a functionalportion thereof. In some embodiments, a complementary affinity moleculeis an avidin-like protein or a derivative or a functional portionthereof, for example but not limited to, rhizavidin or a derivativethereof. In some embodiments, a complementary affinity molecule isavidin or streptavidin or a derivative or a functional portion thereof.

In some embodiments, a polymer of the immunogenic complex is branchedchain polymer, e.g., a branched polysaccharide, or alternatively, can bea straight chain polymer, e.g., a single chain polymer, e.g.,polysaccharide. In some embodiments, the polymer is a polysaccharide,for example, dextran or a derivative thereof. In some embodiments, apolymer, e.g., dextran polysaccharide can be of average molecular weightof 425 kD 500 kDa, inclusive, or in some embodiments, greater than 500kDa. In some embodiments, a polymer, e.g., dextran polysaccharide can beof average molecular weight of 60 kD-90 kDa, inclusive, or in someembodiments, smaller than 70 kDa. The dextran polymer can be derivedfrom a bacterium, such as Leuconostoc mesenteroides.

In some embodiments, an immunogenic composition as disclosed hereincomprises at least 2 antigens, or at 3 least antigens, or at least 5antigens, or between 2-10 antigens, or between 10-15 antigens, orbetween 15-20 antigens, or between 20-50 antigens, or between 50 100antigens, or more than 100 antigens, inclusive. In some embodiments,where an immunogenic composition as disclosed herein comprises at least2 antigens, the antigens can be the same antigen or at least 2 differentantigens. In some embodiments, the antigens can be from the same ordifferent pathogens, or can be different epitopes or parts of the sameantigenic protein, or can be the same antigen which is specific todifferent serotypes or seasonal variations of the same pathogen (e.g.,influenza virus A, B, and C).

A pneumococcal antigen as disclosed herein when present alone, or in animmunogenic complex can elicit both humoral and cellular responses toone or multiple antigens at the same time. The immunogenic compositionsprovide for a long-lasting memory response, potentially protecting asubject from future infection. This allows for a single immunogeniccomposition that raises a high titer of functional anti-polysaccharideantibody, and is similar or compares favorably with the antibody levelinduced by conventional conjugate vaccine

In another embodiment of the present invention, an immunogeniccomposition can comprise a fusion protein polysaccharide conjugate,consisting of a pneumococcal antigen selected from Table 1, fused toPdT, where the pneumococcal antigen:PdT fusion protein is conjugated toa polysaccharide, such that immunity to pneumococcal antigen isenhanced. The polysaccharide may be dextran, a Vi polysaccharide ofSalmonella typhi, or pneumococcal cell wall polysaccharide (CWPS), oranother polysaccharide of prokaryotic or eukaryotic origin.

Nucleic acids encoding truncated and/or mutated forms of a S. pneumoniaeprotein antigens can be fused to nucleotide sequences encoding (1) otherpneumococcal proteins, such as autolysin, surface protein A,neuraminidase, hyaluronate lysate, choline binding protein A, or (2)non-pneumococcal proteins from organisms such as hemophilus influenza b,meningococcus group A, B, or C, or streptococcus group B. The nucleicacids encoding such fused protein are expressed in the expressionsystems.

Pneumolysin truncates can be useful carriers of polysaccharides, ashosts may lacking pre-existing antibodies to such a carrier polypeptide.Pneumolysin is a virulence factor in pneumococcal infections and thereis little antigenic variation of the pneumolysin among pneumococci withdifferent subtypes.

The polysaccharide-protein conjugate, when administered to a mammal suchas a human, induces immune response that exceeds in magnitude, type,and/or duration the immune response induced by administration to amammal of only the polysaccharide component. Accordingly, thepolypeptide component must be of a length sufficient to induce such anenhanced immune response. For fragments of a naturally occurringpneumococcal antigen, the fragments are at least 8, 10, 25, 50, 75, 100,125, 150, 175, 200, 250, 300, 350, 400, 425, 450, 460, 465, 460, 465, ormore amino acids in length. For polypeptides, varying in sequence from anaturally occurring pneumococcal antigens, the polypeptide can be atleast about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% ormore identical to a naturally occurring pneumococcal antigens listed inTable 1.

The polysaccharide component of the conjugate can be any S. pneumoniaecapsular polysaccharide, including but not limited to, any of subtypes1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A,19F, 19A, 20, 22F, 23A, 23F, 24F, 27, 33F, or 34. In some embodiments,the capsular polysaccharide is selected from subtypes 4, 6B, 9V, 14,18C, 19F, or 23F. In some embodiments, the polysaccharide is serotype14. In other embodiments, the polysaccharide is serotype 18C. One ormore of different capsular polysaccharides can be conjugated to a singlepolypeptide or a plurality of polypeptides. For example, a multivalentconjugate can include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10different capsular polysaccharides. Polysaccharides can be conjugated topolypeptides, for example, via a monomeric linkage (only one end of thepolysaccharide is attached to the polypeptide), a looped linkage (asingle polypeptide is attached to looped polysaccharides), orcross-linked (multiple polysaccharides attached to multiplepolypeptides).

Methods for the purification of polypeptides, e.g., pneumococcal antigenpolypeptides described in the examples and in Table 1, and theconjugation of polysaccharides to polypeptides are described in in,e.g., U.S. Pat. Nos. 4,242,501; 4,686,102; 5,623,057; and 5,565,204,which are incorporated herein in their entirety by reference.

The conjugates or polypeptides described herein can be administered to amammal to elicit an immune response (a prophylactic and/or therapeuticimmune response) against S. pneumoniae in the mammal. A pharmaceuticalcomposition containing a conjugate or polypeptide can be delivered in apharmaceutically acceptable carrier, buffer, or preservative which issuitable for a vaccine including, but not limited to physiologicalsaline or other injectable liquids. Additives customary in vaccines mayalso be present, for example stabilizers such as lactose or sorbitol,and adjuvants to enhance the immunogenic response such as aluminumphosphate, hydroxide, or sulphate and stearyl tyrosine. The vaccineproduced may also be used as components of multivalent vaccines whichelicit an immune response against a plurality of infectious agents.

In some embodiments, a pneumococcal polypeptide antigen ispost-translationally modified, e.g. by phosphorylation, myristoylation,acylation, glycosylation, glycation, and the like. In some embodiments,a pneumococcal polypeptide antigen is lipidated. Conjugation to thelipid moiety may be direct or indirect (e.g., via a linker). The lipidmoiety may be synthetic or naturally produced. In some embodiments, apneumococcal polypeptide antigen is chemically conjugated to a lipidmoiety. In some embodiments, a DNA construct encoding a pneumococcalpolypeptide antigen comprises a lipidation sequence. A lipidationsequence may be N-terminal or C-terminal to the polypeptide, and may beembedded in a signal or other sequence. An exemplary lipidation sequenceis the signal sequence of the E. coli gene RlpB.

In some embodiments, a pneumococcal polypeptide antigen is covalentlybound to another molecule. This may, for example, increase thehalf-life, solubility, bioavailability, or immunogenicity of theantigen. Molecules that may be covalently bound to the antigen include acarbohydrate, biotin, poly(ethylene glycol) (PEG), polysialic acid,N-propionylated polysialic acid, nucleic acids, polysaccharides, andPLGA. In some embodiments, the naturally produced form of a polypeptideis covalently bound to a moiety that stimulates the immune system. Anexample of such a moiety is a lipid moiety. In some instances, lipidmoieties are recognized by a Toll-like receptor (TLR) such as TLR2 orTLR4 and activate the innate immune system.

DNA Vaccines; Nucleic Acid Compositions and Antigen Expression

In certain aspects, the vaccine comprises one or more of the nucleicacids disclosed herein or corresponding to the polypeptides describedherein. When a nucleic acid vaccine is administered to a patient, thecorresponding gene product (such as a desired antigen) is produced inthe patient's body. In some embodiments, nucleic acid vaccine vectorsthat include optimized recombinant polynucleotides can be delivered to amammal (including humans) to induce a therapeutic or prophylactic immuneresponse. The nucleic acid may be, for example, DNA, RNA, or a syntheticnucleic acid. The nucleic acid may be single stranded or doublestranded.

Various types of vectors are suitable for expression of pneumococcalantigens in an expression system (e.g., in a host cell). In someembodiments, a composition includes a vector suitable for expression invitro (whether in a cell or in a cell-free system), e.g., for producinga polypeptide composition. The term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked and can include, for example, a plasmid, cosmid or viralvector. The vector can be capable of autonomous replication or it canintegrate into a host DNA. Viral vectors include, e.g., replicationdefective retroviruses, adenoviruses and adeno-associated viruses. Othertypes of viral vectors are known in the art.

Nucleic acid vaccine vectors (e.g., adenoviruses, liposomes,papillomaviruses, retroviruses, etc.) can be administered directly tothe mammal for transduction of cells in vivo. The nucleic acid vaccinescan be formulated as pharmaceutical compositions for administration inany suitable manner, including parenteral administration. Plasmidvectors are typically more efficient for gene transfer to muscle tissue.The potential to deliver DNA vectors to mucosal surfaces by oraladministration has also been reported (PLGA encapsulated Rotavirus andHepatitis B) and DNA plasmids have been utilized for direct introductionof genes into other tissues. DNA vaccines have been introduced intoanimals primarily by intramuscular injection, by gene gun delivery, orby electroporation. After being introduced, the plasmids are generallymaintained episomally without replication. Expression of the encodedproteins has been shown to persist for extended time periods, providingstimulation of B and T cells.

In determining the effective amount of the vector to be administered inthe treatment or prophylaxis of an infection or other condition, thephysician evaluates vector toxicities, progression of the disease, andthe production of anti-vector antibodies, if any. Often, the doseequivalent of a naked nucleic acid from a vector is from about 1 μg to 1mg for a typical 70 kilogram patient, and doses of vectors used todeliver the nucleic acid are calculated to yield an equivalent amount oftherapeutic nucleic acid. Administration can be accomplished via singleor divided doses. The toxicity and therapeutic efficacy of the nucleicacid vaccine vectors can be determined using standard pharmaceuticalprocedures in cell cultures or experimental animals.

A nucleic acid vaccine can contain DNA, RNA, a modified nucleic acid, ora combination thereof. In some embodiments, the vaccine comprises one ormore cloning or expression vectors; for instance, the vaccine maycomprise a plurality of expression vectors each capable of autonomousexpression of a nucleotide coding region in a mammalian cell to produceat least one immunogenic polypeptide. An expression vector oftenincludes a eukaryotic promoter sequence, such as the nucleotide sequenceof a strong eukaryotic promoter, operably linked to one or more codingregions. The compositions and methods herein may involve the use of anyparticular eukaryotic promoter, and a wide variety are known; such as aCMV or RSV promoter. The promoter can be heterologous with respect tothe host cell. The promoter used may be a constitutive promoter.

A vector useful in the present compositions and methods can be circularor linear, single-stranded or double stranded and can be a plasmid,cosmid, or episome. In a suitable embodiment, each nucleotide codingregion is on a separate vector; however, it is to be understood that oneor more coding regions can be present on a single vector, and thesecoding regions can be under the control of a single or multiplepromoters.

Numerous plasmids may be used for the production of nucleic acidvaccines. Suitable embodiments of the nucleic acid vaccine employconstructs using the plasmids VR1012 (Vical Inc., San Diego Calif.),pCMVI.UBF3/2 (S. Johnston, University of Texas) or pcDNA3.1 (InVitrogenCorporation, Carlsbad, Calif.) as the vector. In addition, the vectorconstruct can contain immunostimulatory sequences (ISS), such asunmethylated dCpG motifs, that stimulate the animal's immune system. Thenucleic acid vaccine can also encode a fusion product containing theimmunogenic polypeptide. Plasmid DNA can also be delivered usingattenuated bacteria as delivery system, a method that is suitable forDNA vaccines that are administered orally. Bacteria are transformed withan independently replicating plasmid, which becomes released into thehost cell cytoplasm following the death of the attenuated bacterium inthe host cell.

DNA vaccines, including the DNA encoding the desired antigen, can beintroduced into a host cell in any suitable form including, the fragmentalone, a linearized plasmid, a circular plasmid, a plasmid capable ofreplication, an episome, RNA, etc. Preferably, the gene is contained ina plasmid. In certain embodiments, the plasmid is an expression vector.Individual expression vectors capable of expressing the genetic materialcan be produced using standard recombinant techniques. See e.g.,Maniatis et al., 1985 Molecular Cloning: A Laboratory Manual or DNACloning, Vol. I and II (D. N. Glover, ed., 1985) for general cloningmethods.

Routes of administration include, but are not limited to, intramuscular,intranasal, intraperitoneal, intradermal, subcutaneous, intravenous,intraarterially, intraoccularly and oral as well as topically,transdermally, by inhalation or suppository or to mucosal tissue such asby lavage to vaginal, rectal, urethral, buccal and sublingual tissue.Typical routes of administration include intramuscular, intraperitoneal,intradermal and subcutaneous injection. Genetic constructs may beadministered by means including, but not limited to, traditionalsyringes, needleless injection devices, “microprojectile bombardmentgene guns”, or other physical methods such as electroporation (“EP”),“hydrodynamic method”, or ultrasound. DNA vaccines can be delivered byany method that can be used to deliver DNA as long as the DNA isexpressed and the desired antigen is made in the cell.

In some embodiments, a DNA vaccine is delivered via known transfectionreagents such as cationic liposomes, fluorocarbon emulsion, cochleate,tubules, gold particles, biodegradable microspheres, or cationicpolymers. Cochleae delivery vehicles are stable phospholipid calciumprecipitants consisting of phosphatidyl serine, cholesterol, andcalcium; this nontoxic and noninflammatory transfection reagent can bepresent in a digestive system. Biodegradable microspheres comprisepolymers such as poly(lactide-co-glycolide), a polyester that can beused in producing microcapsules of DNA for transfection. Lipid-basedmicrotubes often consist of a lipid of spirally wound two layers packedwith their edges joined to each other. When a tubule is used, thenucleic acid can be arranged in the central hollow part thereof fordelivery and controlled release into the body of an animal.

In some embodiments, DNA vaccine is delivered to mucosal surfaces viamicrospheres. Bioadhesive microspheres can be prepared using differenttechniques and can be tailored to adhere to any mucosal tissue includingthose found in eye, nasal cavity, urinary tract, colon andgastrointestinal tract, offering the possibilities of localized as wellas systemic controlled release of vaccines. Application of bioadhesivemicrospheres to specific mucosal tissues can also be used for localizedvaccine action. In some embodiments, an alternative approach for mucosalvaccine delivery is the direct administration to mucosal surfaces of aplasmid DNA expression vector which encodes the gene for a specificprotein antigen.

The DNA plasmid vaccines according to the present invention areformulated according to the mode of administration to be used. In someembodiments where DNA plasmid vaccines are injectable compositions, theyare sterile, and/or pyrogen free and/or particulate free. In someembodiments, an isotonic formulation is preferably used. Generally,additives for isotonicity can include sodium chloride, dextrose,mannitol, sorbitol and lactose. In some embodiments, isotonic solutionssuch as phosphate buffered saline are preferred. In some embodiments,stabilizers include gelatin and albumin. In some embodiments, avasoconstriction agent is added to the formulation. In some embodiments,a stabilizing agent that allows the formulation to be stable at room orambient temperature for extended periods of time, such as LGS or otherpolycations or polyanions is added to the formulation.

In some embodiments, the DNA vaccine may further comprises apharmacologically acceptable carrier or diluent. Suitable carriers forthe vaccine are well known to those skilled in the art and include butare not limited to proteins, sugars, etc. Such carriers may be aqueousor non-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous carriers are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose, andthe like. Preservatives and antimicrobials include antioxidants,chelating agents, inert gases and the like. Preferred preservativesinclude formalin, thimerosal, neomycin, polymyxin B and amphotericin B.

An alternative approach to delivering the nucleic acid to an animalinvolves the use of a viral or bacterial vector. Examples of suitableviral vectors include adenovirus, polio virus, pox viruses such asvaccinia, canary pox, and fowl pox, herpes viruses, including catfishherpes virus, adenovirus-associated vector, and retroviruses. Exemplarybacterial vectors include attenuated forms of Salmonella, Shigella,Edwardsiella ictaluri, Yersinia ruckerii, and Listeria monocytogenes. Insome embodiments, the nucleic acid is a vector, such as a plasmid, thatis capable of autologous expression of the nucleotide sequence encodingthe immunogenic polypeptide.

A vector can include a nucleic acid encoding a pneumococcal antigen in aform suitable for expression of the nucleic acid in a host cell. Arecombinant expression vector typically includes one or more regulatorysequences operatively linked to the nucleic acid sequence to beexpressed. Regulatory sequences include promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Regulatorysequences include those which direct constitutive expression of anucleotide sequence, as well as tissue-specific regulatory and/orinducible sequences. A sequence encoding a pneumococcal antigen caninclude a sequence encoding a signal peptide (e.g., a heterologoussignal peptide) such that the antigen is secreted from a host cell. Thedesign of the expression vector can depend on such factors as the choiceof the host cell to be transformed, the level of expression of proteindesired, and the like.

Recombinant expression vectors can be designed for expression andproduction of pneumococcal antigens in prokaryotic or eukaryotic cells.For example, antigens can be expressed in E. coli, insect cells (e.g.,using baculovirus expression vectors), yeast cells or mammalian cells.Suitable host cells are discussed further in Goeddel, Gene ExpressionTechnology Methods in Enzymology 185, Academic Press, San Diego, Calif.,1990. Alternatively, a recombinant expression vector can be transcribedand translated in vitro, for example using T7 promoter regulatorysequences and T7 polymerase.

Expression of polypeptides in prokaryotes is often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein, e.g.,to the amino terminus or carboxy terminus of the recombinant protein,e.g., to increase expression of recombinant protein; to increase thesolubility of the recombinant protein; and/or to aid in the purificationof the recombinant antigen by acting as a ligand in affinitypurification. Often, a proteolytic cleavage site is introduced at thejunction of the fusion moiety and the recombinant antigen to enableseparation of the recombinant antigen from the fusion moiety subsequentto purification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. Gene 67:31-40, 1988), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRITS (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein. Pneumococcalantigen expression vectors provided herein include yeast expressionvectors, vectors for expression in insect cells (e.g., a baculovirusexpression vector) and vectors suitable for expression in mammaliancells.

An expression vector for use in mammalian cells can include viralregulatory elements. For example, commonly used promoters are derivedfrom polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. Avector can include an inducible promoter, e.g., a promoter regulated bya steroid hormone, by a polypeptide hormone (e.g., by means of a signaltransduction pathway), or by a heterologous polypeptide (e.g., thetetracycline-inducible systems, “Tet-On” and “Tet-Off”; see, e.g.,Clontech Inc., CA, Gossen and Bujard, Proc. Natl. Acad. Sci. USA89:5547, 1992, and Paillard, Human Gene Therapy 9:983, 1989).

A host cell can be any prokaryotic or eukaryotic cell. For example, apneumococcal antigen can be expressed in bacterial cells (such as E.coli), insect cells, yeast or mammalian cells (such as Chinese hamsterovary cells (CHO) or COS cells (African green monkey kidney cells CV-1origin SV40 cells; Gluzman, Cell 23:175-182, 1981). Other suitable hostcells are known to those skilled in the art.

Vector DNA can be introduced into host cells via conventionaltransformation or transfection techniques. As used herein, the terms“transformation” and “transfection” are intended to refer to a varietyof art-recognized techniques for introducing foreign nucleic acid (e.g.,DNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, genegun, or electroporation.

A host cell can be used to produce (i.e., express) a pneumococcalantigen. Accordingly, the invention further provides methods forproducing a pneumococcal antigen using host cells. In one embodiment,the method includes culturing a host cell (into which a recombinantexpression vector encoding a pneumococcal antigen has been introduced)in a suitable medium such that a pneumococcal antigen is produced. Inanother embodiment, the method further includes isolating a pneumococcalantigen from the medium or the host cell. Purified pneumococcal antigenscan be used for administration to mammals to induce an immune response,and/or to generate antibodies specific for the antigens.

The present invention also provides nucleic acid compositions thatencode pneumococcal antigens for administration to a subject in vivo,e.g., to elicit an immune response to the antigen. In some embodiments,a nucleic acid composition for administration in vivo includes a nakedDNA plasmid encoding a pneumococcal antigen. Bacterial vectors, repliconvectors, live attenuated bacteria, and viral vectors for expression ofheterologous genes also can be used. Live attenuated viral vectors(e.g., recombinant vaccinia (e.g., modified vaccinia Ankara (MVA), IDTGermany), recombinant adenovirus, avian poxvirus (e.g., canarypox (e.g.,ALVAC™, Aventis Pasteur) or fowlpox), poliovirus, and alphavirus virionvectors) have been successful in inducing cell-mediated immune responseto antigens. Avian poxviruses are defective in mammalian hosts, but canexpress inserted heterologous genes under early promoters. Recombinantadenovirus and poliovirus vectors can thrive in the gut and so canstimulate efficient mucosal immune responses. Finally, attenuatedbacteria can also be used as a vehicle for DNA vaccine delivery.Examples of suitable bacteria include S. enterica, S. tymphimurium,Listeria, and BCG. The use of mutant bacteria with weak cell walls canaid the exit of DNA plasmids from the bacterium.

Nucleic acid compositions used for immunization can include an adjuvant(e.g., an adjuvant such as a polymer, a saponin, muramyl dipeptide,liposomes, immunomodulatory oligonucleotide, or another adjuvantdescribed herein) to promote nucleic acid uptake. Regardless of route,adjuvants can be administered before, during, or after administration ofthe nucleic acid. In some embodiments, an adjuvant increases the uptakeof nucleic acid into host cells and/or increases expression of theantigen from the nucleic acid within the cell, induce antigen-presentingcells to infiltrate the region of tissue where the antigen is beingexpressed, or increase the antigen-specific response provided bylymphocytes.

The terms “homology”, “identity” and “similarity” refer to the degree ofsequence similarity between two peptides or between two optimallyaligned nucleic acid molecules. Homology and identity can each bedetermined by comparing a position in each sequence which can be alignedfor purposes of comparison. For example, it is based upon using astandard homology software in the default position, such as BLAST,version 2.2.14. When an equivalent position in the compared sequences isoccupied by the same base or amino acid, then the molecules areidentical at that position; when the equivalent site occupied by similaramino acid residues (e.g., similar in steric and/or electronic naturesuch as, for example conservative amino acid substitutions), then themolecules can be referred to as homologous (similar) at that position.Expression as a percentage of homology/similarity or identity refers toa function of the number of similar or identical amino acids atpositions shared by the compared sequences, respectively. A sequencewhich is “unrelated” or “non-homologous” shares less than 40% identity,though preferably less than 25% identity with the sequences as disclosedherein.

In some embodiments, a pneumococcal antigen is a variant of apneumococcal antigen of Table 1 which is substantial identical to asequence of SEQ ID NO: 1-76 or SEQ ID NO: 153-234. The term “substantialidentity” as used herein denotes a characteristic of a polynucleotide oramino acid sequence, wherein the polynucleotide or amino acid comprisesa sequence that has at least 85% sequence identity, preferably at least90% to 95% sequence identity, more usually at least 99% sequenceidentity as compared to a reference sequence over a comparison window ofat least 18 nucleotide (6 amino acid) positions, frequently over awindow of at least 24-48 nucleotide (8-16 amino acid) positions, whereinthe percentage of sequence identity is calculated by comparing thereference sequence to the sequence which can include deletions oradditions which total 20 percent or less of the reference sequence overthe comparison window. The reference sequence can be a subset of alarger sequence. The term “similarity”, when used to describe apolypeptide, is determined by comparing the amino acid sequence and theconserved amino acid substitutes of one polypeptide to the sequence of asecond polypeptide.

In some embodiments, a pneumococcal antigen is homologous to a sequenceof SEQ ID NO: 1-76 or SEQ ID NO: 153-234. As used herein, the terms“homologous” or “homologues” are used interchangeably, and when used todescribe a polynucleotide or polypeptide, indicates that twopolynucleotides or polypeptides, or designated sequences thereof, whenoptimally aligned and compared, for example using BLAST, version 2.2.14with default parameters for an alignment (see herein) are identical,with appropriate nucleotide insertions or deletions or amino-acidinsertions or deletions, in at least 60% of the nucleotides, usuallyfrom about 75% to 99%, and more preferably at least about 98 to 99% ofthe nucleotides. The term “homolog” or “homologous” as used herein alsorefers to homology with respect to structure and/or function. Withrespect to sequence homology, sequences are homologs if they are atleast 60 at least 70%, at least 80%, at least 90%, at least 95%identical, at least 97% identical, or at least 99% identical.Determination of homologs of the genes or peptides of the presentinvention can be easily ascertained by the skilled artisan.

In some cases, it there may be advantages to designing vaccines based onantigens that are “surface-expressed” rather than cytoplasmic. Annotatedgenomes often describes protein location based on homology with otheridentified proteins, but this may be an imperfect or often incorrectapproach, as homologous proteins form two different organisms may notnecessarily be located at the same site (nor have the same function) inboth. Thus, an additional tool for determination of the location of theprotein (surface versus other) may be very helpful and may also be usedin the chloroform method described herein.

An embodiment of the present method comprises identifying a protein, “X”of interest, then removing the gene encoding for X from the organism,then replacing that gene with a gene encoding for a tagged version ofthe X protein (e.g., tagged with His, HA, OVA peptide, among others),which can be detected readily with monoclonal or polyclonal antibodies.After confirmation of the genetic construct, the organism is then grown,stained with an antibody that recognizes the tag (and is also fused to afluorophore). Flow cytometry is then used to evaluate whether theantibodies are attached to the surface of the organism, in which case,the antigen can be deduced to be surface-expressed. Similar strategiesusing antibodies attached to magnetic beads can be used as well. Forpneumococcus, for example, the organism can be evaluated in itsencapsulated or unencapsulated form. An antigen can be surfaceexpressed, but hidden under the capsule, for selection of antigenpurposes, it may be advantageous to select an antigen that is bothsurface expressed and accessible despite capsulation.

The identified immunogenic proteins or mixtures thereof may be used in amultivalent or individual vaccine, which can be administered in manyforms (intramuscularly, subcutaneously, mucosally, transdermally). Forexample, combinations or permutations of the twelve pneumococcalimmunogens may be more efficacious against colonization versus disease.A combination of several immunogens with both characteristics mayprovide a superior vaccine.

Antibodies

Antibodies directed against a pneumococcal antigen or functionalfragment thereof as disclosed in Table 1 may be used in a prophylacticor therapeutic application to confer immunity from a first individual toa second individual (e.g., to augment the second individual's immuneresponse against S. pneumoniae or to provide a response if the secondindividual is an immunocompromised patient). Antibodies directed againsta a pneumococcal antigen as disclosed herein can be generated in animmunocompetent host (e.g., by administering to the immunocompetent hosta conjugate described herein), harvested from the host and transfusedinto a recipient in need of treatment or prophylaxis, thereby conferringresistance to the recipient against not only the pneumolysin toxin, butalso against S. pneumoniae and any possibly other bacteria which bindantibodies elicited by the pneumococcal antigen.

Antibodies elicited by a composition described herein can be formulatedas a pharmaceutical composition and be used to confer a prophylactic ortherapeutic immune response to an individual. Suitable components andmethods of administration for pharmaceutical compositions are describedherein. For eliciting passive immunity, the pharmaceutical compositionmay contain polyclonal antibodies or monoclonal antibodies or theirderivatives of fragments. A pharmaceutical composition contains aprophylactically or therapeutically effective amount of an antibody,fragment, or derivative, as determined by standard clinical techniques.

Accordingly, this invention provides, inter alia, antibodies, orantigen-binding fragments thereof, to a novel pneumococcal antigendescribed herein, e.g., those listed in Table 1, or a SP0785 polypeptideantigen, a SP1500 polypeptide antigen, a SP0346 polypeptide antigen, aSP1386 polypeptide antigen, a SP0084 polypeptide antigen, a SP1479polypeptide antigen, a CT067 polypeptide antigen, a CT476 polypeptideantigen, a p6 polypeptide antigen, a SP2145 polypeptide antigen, or anypneumococcal antigen listed in Table 1, e.g., SEQ ID NO: 1-76 or SEQ IDNO: 153-234), or functional fragments or variants thereof. Theantibodies can be of the various isotypes, including: IgG (e.g., IgG1,IgG2, IgG3, IgG4), IgM, IgA1, IgA2, IgD, or IgE. In some embodiments, anantibody is an IgG isotype, e.g., IgG1. An antibody against apneumococcal antigen can be full-length (e.g., an IgG1 or IgG4 antibody)or can include only an antigen-binding fragment (e.g., a Fab, F(ab)₂, Fvor a single chain Fv fragment). These include monoclonal antibodies,recombinant antibodies, chimeric antibodies, human antibodies, andhumanized antibodies, as well as antigen-binding fragments of theforegoing.

Monoclonal antibodies can be produced by a variety of techniques,including conventional monoclonal antibody methodology, e.g., thestandard somatic cell hybridization technique of Kohler and Milstein,Nature 256: 495, 1975. Polyclonal antibodies can be produced byimmunization of animal or human subjects. See generally, Harlow, E. andLane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N. Y., 1988. Antibodies against pneumococcalantigens described herein can be used, e.g., for diagnostic assays, orfor therapeutic applications.

In some embodiments of the present invention, a subject's response to animmunogenic composition described herein is evaluated, e.g., todetermine efficacy of the composition, and/or to compare responseselicited by the composition to responses elicited by a differentcomposition.

Nucleic Acids Encoding Pneumococcal Polypeptide Antigens

Nucleic acids encoding a pneumococcal polypeptide antigen or a fragmentor variant of pneumococcal polypeptide can be administered to a mammal(e.g., a human) to generate a prophylactic and/or therapeutic immuneresponse in the mammal. The immune response can be an anti-pneumococcalhumoral and/or a cellular immune response.

Polypeptides that can be encoded by the nucleic acid constructs includeone or more pneumococcal polypeptide antigens, alone or fused to afusion polypeptide, e.g. PdT and/or a polysaccharide and/or anotherantigen, e.g., Vi. In addition, a nucleic acid can encode a combinationof two or more such polypeptides, fragments, or variants.

Nucleic acid expression constructs can be prepared by using standardrecombinant DNA methods. Regulatory elements can be included in aconstruct to facilitate expression of the nucleic acid encoding thepolypeptide. These elements include sequences for enhancing expressionin human or other mammalian cells, e.g., promoters, RNA stabilizationsequences 5′ and/or 3′ to the coding sequence, introns (which can beplaced at any location within or adjacent to the encoded sequence), andpoly(A) addition sites, as well as an origin of replication and one ormore genes encoding selectable markers enabling the constructs toreplicate and be selected in prokaryotic and/or eukaryotic hosts. A T7polymerase promoter or other type of promoter (e.g., a tissue-specificpromoter or a cell-specific promoter such as a muscle-specific promoter)is optionally present at the 5′ end of the coding sequence, and asequence encoding a FLAG or other mAb determinant is optionally presentat the 3′ end of the coding sequence. The construct may also containother transcriptional and translational signals, such as a Kozaksequence.

The construct may in addition include a sequence encoding a targetingsignal that directs the encoded polypeptide to a desired intracellularcompartment, the targeting signal being linked to the polypeptide.Targeting signals can direct the encoded polypeptide to endoplasmicreticulum (ER), the golgi, the nucleus, a lysosome, a class II peptideloading compartment, or an endosome, and include signal peptides, ERretention peptides, and lysosome-targeting peptides.

The nucleic acids can be used in any vector that allows for expressionin cells of a mammal. The vector can be, e.g., a non-viral vector suchas a plasmid or bacterial vector, an integrating viral vector, or anon-integrating viral vector. An example of a suitable vector is thefamily of pcDNA mammalian expression vectors (Invitrogen), which permitdirect and rapid cloning of PCR products.

Various delivery systems can be used to deliver nucleic acids encodingpolypeptides into appropriate cells. The nucleic acids encoding thepolypeptides can be delivered in a pharmaceutically acceptable carriersuch as saline, or as colloidal suspensions, or as powders, with orwithout diluents. The nucleic acids can be “naked” or associated withdelivery vehicles and delivered using delivery systems known in the art,such as lipids, liposomes, microspheres, microparticles ormicrocapsules, gold particles, ISCOMS, nanoparticles, polymers,condensing agents, polysaccharides, polyamino acids, dendrimers,saponins, QS21, adsorption enhancing materials, adjuvants, or fattyacids. Nucleic acids can also be delivered to a cell, e.g., a skeletalmuscle cell, either in vitro or in vivo, using electroporation.

The nucleic acids can be administered using standard methods, e.g.,those described in Donnelly et al., J. Immunol Methods 176:145, 1994,and Vitiello et al., J. Clin. Invest. 95:341, 1995, and can be deliveredinto subjects in any manner known in the art, e.g., orallyintramuscularly, intravenously, intraarterially, intrathecally,intradermally, intraperitoneally, intranasally, intrapulmonarily,intraocularly, intravaginally, intrarectally or subcutaneously. They canbe introduced into the gastrointestinal tract or the respiratory tract,e.g., by inhalation of a solution or powder containing the nucleicacids. Administration can be local or systemic.

It is expected that a dosage of approximately 100-2000 μg of nucleicacid would be administered to an individual. Where the patient is anadult human, vaccination regimens can include, e.g., intramuscular,intradermal, inhalation, or subcutaneous administrations of 10-1000 μgof a plasmid DNA when delivered in a microparticle, or of about 10-2500μg, e.g., 100 to 2000, or 500 to 1000 μg, of naked plasmid DNA deliveredintramuscularly or intradermally, repeated 3-6 times. As is well knownin the medical arts, dosage for any given patient depends upon manyfactors, including the patient's size, general health, sex, body surfacearea, age, the particular compound to be administered, time and route ofadministration, and other drugs being administered concurrently.Determination of optimal dosage is well within the abilities of apharmacologist of ordinary skill.

Other standard delivery methods, e.g., biolistic transfer or ex vivotreatment, can also be used. In ex vivo treatment, antigen presentingcells (APCs) such as dendritic cells, peripheral blood mononuclearcells, or bone marrow cells can be obtained from a patient or anappropriate donor and activated ex vivo with the nucleic acid, and thenimplanted or reinfused into the patient.

The nucleic acids can be administered alone or in combination with othertherapies known in the art, e.g., antimicrobial agents. In addition, thenucleic acids can be administered in combination with other treatmentsdesigned to enhance immune responses, e.g., by co-administration withadjuvants, cytokines (or nucleic acids encoding cytokines), or CpGoligonucleotides, as is well known in the art.

The ability of a nucleic acid to elicit an immune response in a hostmammal can be assayed by using methods for measuring immune responsesthat are well known in the art. For example, the generation of cytotoxicT cells can be demonstrated in a standard .sup.51Cr release assay, bymeasuring intracellular cytokine expression or secretion, or by usingMHC tetramers. Standard assays, such as ELISA or ELISPOT, can be used tomeasure cytokine profiles attributable to T cell activation. T cellproliferation can be measured using assays such as .sup.3H-thymidineuptake and other assays known in the art. B cell responses can bemeasured using art recognized assays such as ELISA. Other methodologiescan also be used to evaluate the effects of the nucleic acids onpathogen-associated lesions or on other pathogen levels generally (e.g.,pneumococci clearance in challenged mice treated with the conjugate).

The nucleic acids described herein can be used in the manufacture of amedicament for the prevention or treatment of an infection with S.pneumoniae or conditions associated with such infection.

Assays for T Cell Activation

In some embodiments, various assays can be utilized in order tocharacterize an antigen or composition and/or to determine whether animmune response has been stimulated in a T cell or group of T cells. Insome embodiments, assays are used to characterize a T cell response in asubject that has been administered an immunogenic composition to elicitan anti-pneumococcal response (e.g., to evaluate whether a detectable Tcell response has been elicited and/or to evaluate the potency of theresponse). The novel pneumococcal antigens described herein also providediagnostic agents to evaluate exposure to pneumococcal infections (e.g.,in non-vaccinated subjects). In some embodiments, assays are used tocharacterize a T cell response in a subject to determine whether thesubject has been infected with a pneumococcal organism. The subject canbe a subject suspected of exposure to a pneumococcal organism recently(i.e., an assay to detect a response can be performed with a sampletaken from the subject about 3, 4, 5, 6, 7, 8, 9, 10, 14, 30, or moredays after suspected exposure to apneumococcalorganism). The subject canbe a subject suspected of exposure to a pneumococcal organism weeks,months, or years prior to the assay. The novel pneumococcal antigensdescribed herein also provide prognostic agents to evaluate outcomes ofexposure to a pneumococcal organism (e.g., in subjects known to be, orto have been, infected with a pneumococcal organism). In someembodiments, assays are used to characterize a T cell response in asubject to assess the likelihood of sequelae (e.g., sepsis) to infectionwith a pneumococcal organism.

In some embodiments, stimulation of an immune response in T cells isdetermined by measuring antigen-induced production of cytokines by Tcells. In some embodiments, stimulation of an immune response in T cellscan be determined by measuring antigen-induced production of IFN-γ,IL-4, IL-2, IL-6, IL-10, IL-17 and/or TNF-α by T cells. In someembodiments, antigen-induced production of cytokines by T cells can bemeasured by intracellular cytokine staining followed by flow cytometry.Other suitable methods include surface capture staining followed by flowcytometry, or methods that determine cytokine concentration insupernatants of activated T cell cultures, such as ELISA or ELISPOTassays.

In some embodiments, antigen-produced production of cytokines by T cellsis measured by ELISPOT assay. ELISPOT assays typically employ atechnique very similar to the sandwich enzyme-linked immunosorbent assay(ELISA) technique. An antibody (e.g. monoclonal antibody, polyclonalantibody, etc.) is coated aseptically onto a PVDF (polyvinylidenefluoride)-backed microplate. Antibodies are chosen for their specificityfor the cytokine of interest. The plate is blocked (e.g., with a serumprotein that is non-reactive with any of the antibodies in the assay).Cells to be tested for cytokine production are plated out at varyingdensities, along with antigen or mitogen, and then placed in ahumidified 37° C. CO₂ incubator for a specified period of time. Cytokinesecreted by activated cells is captured locally by the coated antibodyon the high surface area PVDF membrane. After washing the wells toremove cells, debris, and media components, a secondary antibody (e.g. abiotinylated polyclonal antibody) specific for the cytokine is added tothe wells. This antibody is reactive with a distinct epitope of thetarget cytokine and thus is employed to detect the captured cytokine.Following a wash to remove any unbound biotinylated antibody, thedetected cytokine is then visualized using an avidin-HRP, and aprecipitating substrate (e.g., AEC, BCIP/NBT). The colored end product(a spot, usually red or blue) typically represents an individualcytokine-producing cell. Spots can be counted manually (e.g., with adissecting microscope) or using an automated reader to capture themicrowell images and to analyze spot number and size. In someembodiments, each spot correlates to a single cytokine-producing cell.

In some embodiments, an immune response in T cells is said to bestimulated if between about 1% and about 100% of antigen-specific Tcells produce cytokines. In some embodiments, an immune response in Tcells is said to be stimulated if at least about 1%, at least about 5%,at least about 10%, at least about 25%, at least about 50%, at leastabout 75%, at least about 90%, at least about 95%, at least about 99%,or about 100% of antigen-specific T cells produce cytokines.

In some embodiments, an immune response in T cells is said to bestimulated if immunized subjects comprise at least about 10-fold, atleast about 50-fold, at least about 100-fold, at least about 500-fold,at least about 1000-fold, at least about 5000-fold, at least about10.000-fold, at least about 50.000-fold, at least about 100.000-fold, orgreater than at least about 100.000-fold more cytokine-producing cellsthan do naive controls.

In some embodiments, stimulation of an immune response in T cells can bedetermined by measuring antigen-induced proliferation of T cells. Insome embodiments, antigen-induced proliferation may be measured asuptake of H₃-thymidine in dividing T cells (sometimes referred to as“lymphocyte transformation test, or “LTT”). In some embodiments,antigen-induced proliferation is said to have occurred if.sup.3H-thymidine uptake (given as number of counts from a .gamma.counter) is at least about 5-fold, at least about 10-fold, at leastabout 20-fold, at least about 50-fold, at least about 100-fold, at leastabout 500-fold, at least about 1000-fold, at least about 5000-fold, atleast about 10.000-fold, or greater than at least about 10.000-foldhigher than a naive control.

In some embodiments, antigen-induced proliferation may be measured byflow cytometry. In some embodiments, antigen-induced proliferation maybe measured by a carboxyfluorescein succinimidyl ester (CFSE) dilutionassay. CFSE is a non-toxic, fluorescent, membrane-permeating dye thatbinds the amino groups of cytoplasmic proteins with itssuccinimidyl-reactive group (e.g., T cell proteins). When cells divide,CFSE-labeled proteins are equally distributed between the daughtercells, thus halving cell fluorescence with each division. Consequently,antigen-specific T cells lose their fluorescence after culture in thepresence of the respective antigen (CFSE.sup.low) and aredistinguishable from other cells in culture (CFSE.sup.high). In someembodiments, antigen-induced proliferation is said to have occurred ifCFSE dilution (given as the percentage of CFSE^(low) cells out of allCFSE cells) is at least about 5%, at least about 10%, at least about25%, at least about 50%, at least about 75%, at least about 90%, atleast about 95%, or at least about 100%.

In some embodiments, an immune response in T-cells is said to bestimulated if cellular markers of T cell activation are expressed atdifferent levels (e.g., higher or lower levels) relative to unstimulatedcells. In some embodiments, CD11a, CD27, CD25, CD40L, CD44, CD45RO,and/or CD69 are more highly expressed in activated T cells than inunstimulated T cells. In some embodiments, L-selectin (CD62L), CD45RA,and/or CCR7 are less highly expressed in activated T cells than inunstimulated T cells.

In some embodiments, an immune response in T cells is measured byassaying cytotoxicity by effector CD8+ T cells against antigen-pulsedtarget cells. For example, a ⁵¹chromium (⁵¹Cr) release assay can beperformed. In this assay, effector CD8+ T cells bind infected cellspresenting virus peptide on class I MHC and signal the infected cells toundergo apoptosis. If the cells are labeled with ⁵¹Cr before theeffector CD8+ T cells are added, the amount of ⁵¹Cr released into thesupernatant is proportional to the number of targets killed. In someembodiments, an immune response in T cells is measured by an in vivocytotoxicity assay in which target cells are antigen pulsed and labeledwith a fluorescent dye, then transferred into immunized animals.Specific cytolytic T cells cause the disappearance of fluorescentlylabeled cells that are pulsed with a relevant antigen, but no decreasein cells pulsed with a control antigen. See, e.g., Coligan et al.,Current Protocols in Immunology, 3.11.14-16, John Wiley & Sons, Inc.,2007. In some embodiments, an immune response in T cells is measured bydetecting expression of one or more of Perforin, Granzyme B, or CD107a(e.g., by ELISPOT or flow cytometry). See, e.g., Betts et al., J.Immunol Meth. 281(1-2):65-78, 2003.

In Vivo Assays

In some embodiments, an immunogenic composition may be characterized(e.g., to assess efficacy in inducing a beneficial response in animalmodels) by infecting groups of immunized and non-immunized mice (e.g., 3or more weeks after vaccination) with a dose of a chlamydia organismthat typically produces a particular pathology (e.g., upper urogenitaltract infection) or bacterial burden. The magnitude and duration ofpathology or bacterial burden due to infection of both groups ismonitored and compared. In one example, B cell responses arecharacterized by transferring serum from immune mice as a “passivevaccine” to assess protection of non-immune mice from pathologicaleffects or burden of infection. In some embodiments, infiltratingleukocyte populations are characterized (e.g., to assess the number andtype cells in a region of infection, e.g., whether CD4.sup.+ T cells,CD8.sup.+ T cells, or other cell types are present). Animal models forchlamydial urogenital infection have been described. In someembodiments, a chlamydia organism is applied as an intravaginalinoculum, and infection and pathology of one or more of lower and uppergenital tracts of the infected animal is characterized. See, e.g.,Barron et al. (J. Infect. Dis. 143(1):63-6, 1981), which describes anintravaginal infection model in mice. In some embodiments, clearance ofprimary infection is a measure of protective immunity in this model. Insome embodiments, detection of CD4+ T cell responses of a Th1 subtypecorrelate with protection (Morrison et al., Infect Immun 70:2741-2751,2002).

In some embodiments, an immunogenic composition is assessed in an animalmodel of pneumococcal infection. In some embodiments, lower urogenitaltract infection by pneumococcus is assessed in the model (e.g., lowertract bacterial burden and/or inflammation due to infection isassessed). In some embodiments, upper tract infection by chlamydia isassessed in the model (e.g., one or more of upper tract bacterialburden, inflammation, infertility, collagen deposition, scarring due toinfection, are assessed). In some embodiments, an ability to preventascension of a chlamydia infection from the lower tract to the uppergenital tract is assessed. In some embodiments, rate of bacterialclearance from the lower tract is assessed. In some embodiments, rate ofbacterial clearance from the upper tract is assessed. In someembodiment, the rate of bacterial clearance from the nose is assessed.In some embodiments, an immunogenic composition is assessed in an animalmodel in multiple strains of the animal of interest (e.g., multiplemouse strains). In some embodiments, presence and size of hydrosalpinx(fluid blockage of fallopian tubes) is assessed.

In some embodiments, desirable immunogenic compositions arecharacterized as having one or more of the above effects in vivo (e.g.,in an animal model). For example, in some embodiments, an immunogeniccomposition reduces lower urogenital tract infection by chlamydiabacteria. In some embodiments, an immunogenic composition reduces lowertract bacterial burden. In some embodiments, an immunogenic compositionreduces lower tract inflammation due to infection. In some embodiments,an immunogenic composition reduces upper tract infection bypneumococcus. In some embodiments, an immunogenic composition reducesascension of a pneumococcal infection from the lower tract to the uppergenital tract. In some embodiments, an immunogenic composition increasesthe rate of bacterial clearance from the lower tract and/or the uppertract. In some embodiments, an immunogenic composition has one or moreof the above effects in multiple animal strains (e.g., multiple mousestrains).

In some embodiments, pneumococcal antigens of Table 1 or functionalfragments thereof for use in the immunogenic compositions as disclosedherein elicits an IL-17A response in mouse splenocytes or human PBMCs invitro as disclosed in Example 2, and/or protects colonization in vivo ina mouse colonization model, where mice are challenged with a serotype ofpneumococcal strain (e.g., serotypes 6B, 14F or 19F) after biweeklyimmunization with a pneumococcal antigen (or functional fragment), andassessing for presence of pneumococcal colonization in the nose, asdisclosed in Example 3.

One of ordinary skill in the art will recognize that the assaysdescribed above are only exemplary methods which could be utilized inorder to determine whether T cell activation and/or B cell activationhas occurred. Any assay known to one of skill in the art which can beused to determine whether T and/or B cell activation has occurred fallswithin the scope of this invention. The assays described herein as wellas additional assays that could be used to determine whether T and/or Bcell activation has occurred are described in Current Protocols inImmunology (John Wiley & Sons, Hoboken, N. Y., 2007; incorporated hereinby reference).

Ongoing studies assess immunogenicity in outbred animal strains, andcharacterize the efficacy of these proteins in protecting againstinvasive disease in aspiration/sepsis models. In some embodiments, onecan test the ability of a pneumococcal antigen, e.g., an antigen listedin Table 1, or a homologue or functional fragment or variant of anantigen listed in Table 1 to elicit an immune response in vivo, bymeasuring a CMI response to the target antigen. CMI assays are known inthe art and described, for example, in United States Patent Application2005/0014205, WO/1987/005400, U.S. Pat. No. 5,674,698 and commerciallyavailable kits such as IMMUNKNOW® CYLEX Immune cell function assayProduct No. 4400, which are incorporated in their entirety by referenceherein for use in the present invention.

Applications and Use of the Immunogenic Compositions

The S. pneumoniae vaccines described herein may be used for prophylacticand/or therapeutic treatment of S. pneumoniae. Accordingly, thisapplication provides a method for treating a subject suffering from orsusceptible to S. pneumoniae infection, comprising administering aneffective amount of any of the vaccine formulations described herein. Insome aspects, the method inhibits S. pneumoniae colonization in anindividual. In some aspects, the method inhibits S. pneumoniae symptomsor sequelae, such as sepsis. The subject receiving the vaccination maybe a male or a female, and may be a child or adult. In some embodiments,the subject being treated is a human. In other embodiments, the subjectis a non-human animal.

Accordingly, the immunogenic compositions and methods described hereincan be used for the prophylaxis and/or treatment of any pneumococcalinfection, pneumococcal disease, disorder, and/or condition, or apneumococcal infection, such as sepsis. As used herein, “prophylaxis”refers to uses before onset of symptoms due to a pneumococcal infection,pneumococcal disease, disorder, and/or condition and/or before knownexposure to a Streptococcus pneumoniae organism. Subjects include, butare not limited to, humans and/or other primates; and other animalssusceptible to infection by S. pneumoniae organisms, includingcommercially relevant mammals such as cattle, pigs, horses, sheep, cats,and/or dogs; and/or birds, including commercially relevant birds such aschickens, ducks, geese, and/or turkeys.

1. Prophylactic Use

In prophylactic embodiments, a vaccine or immunogenic composition asdisclosed herein is administered to a subject to induce an immuneresponse that can help protect against the establishment of S.pneumoniae, for example by protecting against colonization, the firstand necessary step in disease. Thus, in some aspects, the methodinhibits infection by S. pneumoniae in a non-colonized or uninfectedsubject. In another aspect, the method may reduce the duration ofcolonization in an individual who is already colonized.

In some embodiments, a vaccine or immunogenic composition as disclosedherein confer protective immunity, allowing a vaccinated individual toexhibit delayed onset of symptoms or sequelae, or reduced severity ofsymptoms or sequelae, as the result of his or her exposure to thevaccine. In certain embodiments, the reduction in severity of symptomsor sequelae is at least 25%, 40%, 50%, 60%, 70%, 80% or even 90%. Inparticular embodiments, vaccinated individuals may display no symptomsor sequelae upon contact with S. pneumoniae, do not become colonized byS. pneumoniae, or both. Protective immunity is typically achieved by oneor more of the following mechanisms: mucosal, humoral, or cellularimmunity Mucosal immunity is primarily the result of secretory IgA(sIGA) antibodies on mucosal surfaces of the respiratory,gastrointestinal, and genitourinary tracts. The sIGA antibodies aregenerated after a series of events mediated by antigen-processing cells,B and T lymphocytes, that result in sIGA production by B lymphocytes onmucosa-lined tissues of the body. Humoral immunity is typically theresult of IgG antibodies and IgM antibodies in serum. Cellular immunitycan be achieved through cytotoxic T lymphocytes or through delayed-typehypersensitivity that involves macrophages and T lymphocytes, as well asother mechanisms involving T cells without a requirement for antibodies.In particular, cellular immunity may be mediated by TH1 or TH17 cells.

Essentially any individual has a certain risk of becoming infected withS. pneumoniae. However, certain sub-populations have an increased riskof infection. In some embodiments, a vaccine formulation as describedherein (e.g., a composition comprising one or more polypeptides fromTable 1 or 2, or nucleic acids encoding the polypeptides, or antibodiesreactive with the polypeptides) is administered to patients that areimmunocompromised.

An immunocompromising condition arising from a medical treatment islikely to expose the individual in question to a higher risk ofinfection with S. pneumoniae. It is possible to treat an infectionprophylactically in an individual having the immunocompromised conditionbefore or during treatments known to compromise immune function. Byprophylactically treating with an antigenic composition (e.g., two ormore antigens from Table 1 or 2, or nucleic acids encoding theantigens), or with antibodies reactive to two or more antigens fromTable 1 or 2, before or during a treatment known to compromise immunefunction, it is possible to prevent a subsequent S. pneumoniae infectionor to reduce the risk of the individual contracting an infection due tothe immunocompromised condition. Should the individual contract an S.pneumoniae infection e.g., following a treatment leading to animmunocompromised condition it is also possible to treat the infectionby administering to the individual an antigen composition.

The following groups are at increased risk of pneumococcal disease orits complications, and therefore it is advantageous for subjects fallinginto one or more of these groups to receive a vaccine formulationdescribed herein: children, especially those from 1 month to 5 years oldor 2 months to 2 years old; children who are at least 2 years of agewith asplenia, splenic dysfunction or sickle-cell disease; children whoare at least 2 years of age with nephrotic syndrome, chroniccerebrospinal fluid leak, HIV infection or other conditions associatedwith immunosuppression.

In another embodiment, at least one dose of the pneumococcal antigencomposition is given to adults in the following groups at increased riskof pneumococcal disease or its complications: all persons 65 years ofage; adults with asplenia, subjects with Job's syndrome (subject lackingTh-17 cell-mediated response), subjects with aggamaglobunemia (a subjectlacking antibody-mediated response), splenic dysfunction or sickle-celldisease; adults with the following conditions: chronic cardiorespiratorydisease, cirrhosis, alcoholism, chronic renal disease, nephroticsyndrome, diabetes mellitus, chronic cerebrospinal fluid leak, HIVinfection, AIDS and other conditions associated with immunosuppression(Hodgkin's disease, lymphoma, multiple myeloma, immunosuppression fororgan transplantation), individuals with cochlear implants; individualswith long-term health problems such as heart disease and lung disease,as well as individuals who are taking any drug or treatment that lowersthe body's resistance to infection, such as long-term steroids, certaincancer drugs, radiation therapy; Alaskan natives and certain NativeAmerican populations.

2. Therapeutic Use

In therapeutic applications, a vaccine or immunogenic composition asdisclosed herein may be administered to a patient suffering from S.pneumoniae infection, in an amount sufficient to treat the patient.Treating the patient, in this case, refers to reducing S. pneumoniaesymptoms and/or bacterial load and/or sequelae bin an infectedindividual. In some embodiments, treating the patient refers to reducingthe duration of symptoms or sequelae, or reducing the intensity ofsymptoms or sequelae. In some embodiments, the vaccine reducestransmissibility of S. pneumoniae from the vaccinated patient. Incertain embodiments, the reductions described above are at least 25%,30%, 40%, 50%, 60%, 70%, 80% or even 90%.

In therapeutic embodiments, the vaccine is administered to an individualpost-infection. The vaccine may be administered shortly after infection,e.g. before symptoms or sequelae manifest, or may be administered duringor after manifestation of symptoms or sequelae.

A therapeutic S. pneumoniae vaccine can reduce the intensity and/orduration of the various symptoms or sequelae of S. pneumoniae infection.Symptoms or sequelae of S. pneumoniae infection can take many forms.Invasive pneumococcal diseases include acute sinusitis, otitis media,meningitis, bacteremia, sepsis, osteomyelitis, septic arthritis,endocarditis, peritonitis, pericarditis, cellulitis, and brain abscess.Accordingly, in some cases, an infected patient develops pneumonia,acute sinusitis, otitis media (ear infection), meningitis, bacteremia,sepsis, osteomyelitis, septic arthritis, endocarditis, peritonitis,pericarditis, cellulitis, or brain abscess.

Sepsis is a rare but life-threatening complication of S. pneumoniaeinfection, where the bacterium invades the bloodstream and systemicinflammation results. Typically, fever is observed and white blood cellcount increases. A further description of sepsis is found in Goldstein,B. et al. “International pediatric sepsis consensus conference:definitions for sepsis and organ dysfunction in pediatrics.” PediatrCrit. Care Med. January 2005; 6 (1):2-8.

In some embodiments, immunogenic compositions in accordance with thepresent invention may be used to treat, alleviate, ameliorate, relieve,delay onset of, inhibit progression of, reduce risk of infection by, andreduce severity of, and/or reduce incidence of one or more symptoms orfeatures of a pneumococcal disease, disorder, and/or condition. In someembodiments, inventive an immunogenic composition may be used to treat,alleviate, ameliorate, relieve, delay onset of, inhibit progression of,reduce severity of, and/or reduce incidence of one or more symptoms orfeatures of pneumococcal infection (e.g., but not limited to sepsis,pneumonia, acute otitis media, bacteremia and bacterial meningitis).

In one aspect of the invention, a method for the prophylaxis and/ortreatment of pneumococcal infection is provided. In some embodiments,the prophylaxis and/or treatment of pneumococcal infection comprisesadministering a therapeutically effective amount of an immunogeniccomposition described herein to a subject in need thereof, in suchamounts and for such time as is necessary to achieve the desired result.In certain embodiments of the present invention a “therapeuticallyeffective amount” of an inventive immunogenic composition is that amounteffective for reducing risk of infection by, or treating, alleviating,ameliorating, relieving, delaying onset of, inhibiting progression of,reducing severity of, and/or reducing incidence of one or more symptomsor features of pneumococcal infection. A therapeutically effectiveamount may be determined on a population basis, and is not required tobe an amount that naturally induces a protective response in aparticular subject.

In some embodiments, inventive prophylactic and/or therapeutic protocolsinvolve administering a therapeutically effective amount of one or moreinventive immunogenic compositions to a healthy subject (i.e., a subjectwho does not display any symptoms of chlamydia infection and/or who hasnot been diagnosed with pneumococcal infection). For example, healthyindividuals may be vaccinated using inventive immunogenic compositionsprior to development of pneumococcal infection and/or onset of symptomsof pneumococcal infection; at risk individuals (e.g., patients exposedto individuals suffering from pneumococcal infection, patients with HIVor a compromised immune system, or a subject who is susceptible is topneumococcal infection such as a subject with Job's syndrome (subjectlacking Th-17 cell-mediated response) or a subject with aggamaglobunemic(a subject lacking antibody-mediated response)) can be treatedsubstantially contemporaneously with (e.g., within 48 hours, within 24hours, or within 12 hours of) the onset of symptoms of and/or exposureto pneumococcal infection. Of course individuals known to havepneumococcal infection or sepsis may receive treatment at any time.

In some embodiments, inventive prophylactic and/or therapeutic protocolsinvolve administering a therapeutically effective amount of one or moreinventive immunogenic compositions to a subject such that an immuneresponse is stimulated in both T cells and B cells.

In some embodiments, by combining one or more pneumococcal antigens andadjuvants, immune responses (e.g. T cell and/or B cell responses) can betailored to preferentially elicit the most desirable type of immuneresponse for a given indication, e.g., humoral response, Th1 T cellresponse, Th17 T cell response, IFN-γ secretion by antigen-specific Tcells, cytotoxic T cell response, antibody response, B cell response,innate immune response, or a combination of these responses.

Assaying Vaccination Efficacy

The efficacy of vaccination with the a vaccine or immunogeniccomposition as disclosed herein may be determined in a number of ways,in addition to the clinical outcomes described above. First, one mayassay IL-17 levels (particularly IL-17A) by stimulating T cells derivedfrom the subject after vaccination. The IL-17 levels may be compared toIL-17 levels in the same subject before vaccination. Increased IL-17(e.g., IL-17A) levels, such as a 1.5 fold, 2-fold, 5-fold, 10-fold,20-fold, 50-fold or 100-fold or more increase, would indicate anincreased response to the vaccine. Alternatively (or in combination),one may assay neutrophils in the presence of T cells or antibodies fromthe patient for pneumococcal killing. Increased pneumococcal killing,such as a 1.5 fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or100-fold or more increase, would indicate an increased response to thevaccine. In addition, one may measure T.sub.H17 cell activation, whereincreased T.sub.H17 cell activation, such as a 1.5 fold, 2-fold, 5-fold,10-fold, 20-fold, 50-fold or 100-fold or more increase, correlates withan increased response to the vaccine. One may also measure levels of anantibody specific to the vaccine, where increased levels of the specificantibody, such as a 1.5 fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-foldor 100-fold or more increase, are correlated with increased vaccineefficacy. In certain embodiments, two or more of these assays are used.For example, one may measure IL-17 levels and the levels ofvaccine-specific antibody. Alternatively, one may follow epidemiologicalmarkers such as incidence of, severity of, or duration of pneumococcalinfection in vaccinated individuals compared to unvaccinatedindividuals.

Vaccine efficacy may also be assayed in various model systems such asthe mouse model. For instance, BALB/c or C57BL/6 strains of mice may beused. After administering the test vaccine to a subject (as a singledose or multiple doses), the experimenter administers a challenge doseof S. pneumoniae. In some cases, a challenge dose administeredintranasally is sufficient to cause S. pneumoniae colonization(especially nasal colonization) in an unvaccinated animal, and in somecases a challenge dose administered via aspiration is sufficient tocause sepsis and a high rate of lethality in unvaccinated animals. Onecan then measure the reduction in colonization or the reduction inlethality in vaccinated animals. As demonstrated in Examples 1-3, theefficacy of polypeptides of Table 1 in inhibiting S. pneumoniae nasalcolonization following intranasal challenge in the mouse model can beassessed. Examples 3 and 4 show the efficacy of polypeptides of Table 1in protecting against sepsis and death following infection with S.pneumoniae via aspiration in the mouse model.

Immunogenic Compositions and Formulations

In one embodiment, an immunogenic composition as disclosed herein, e.g.,a vaccine or immunogenic composition described herein, can comprise apharmaceutically acceptable carrier. In another embodiment, the vaccinecomposition described herein is formulated for administering to amammal. Suitable formulations can be found in Remington's PharmaceuticalSciences, 16th and 18th Eds., Mack Publishing, Easton, Pa. (1980 and1990), and Introduction to Pharmaceutical Dosage Forms, 4th Edition, Lea& Febiger, Philadelphia (1985), each of which is incorporated herein byreference.

In one embodiment, a vaccine compositions as described herein comprisepharmaceutically acceptable carriers that are inherently nontoxic andnontherapeutic. Examples of such carriers include ion exchangers,alumina, aluminum stearate, lecithin, serum proteins, such as humanserum albumin, buffer substances such as phosphates, glycine, sorbicacid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts, or electrolytes such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, and polyethyleneglycol. For all administrations, conventional depot forms are suitablyused. Such forms include, for example, microcapsules, nano-capsules,liposomes, plasters, inhalation forms, nose sprays, sublingual tablets,and sustained release preparations. For examples of sustained releasecompositions, see U.S. Pat. Nos. 3,773,919, 3,887,699, EP 58,481A, EP158,277A, Canadian Patent No. 1176565; U. Sidman et al., Biopolymers22:547 (1983) and R. Langer et al., Chem. Tech. 12:98 (1982). Theproteins will usually be formulated at a concentration of about 0.1mg/ml to 100 mg/ml per application per patient.

In one embodiment, other ingredients can be added to vaccineformulations, including antioxidants, e.g., ascorbic acid; low molecularweight (less than about ten residues) polypeptides, e.g., polyarginineor tripeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids, such as glycine, glutamic acid, aspartic acid, or arginine;monosaccharides, disaccharides, and other carbohydrates includingcellulose or its derivatives, glucose, mannose, or dextrins; chelatingagents such as EDTA; and sugar alcohols such as mannitol or sorbitol.

In one embodiment, a vaccine composition as described herein foradministration must be sterile. Sterility is readily accomplished byfiltration through sterile filtration membranes (e.g., 0.2 micronmembranes).

In some embodiments, a vaccine composition as described herein furthercomprises pharmaceutical excipients including, but not limited tobiocompatible oils, physiological saline solutions, preservatives,carbohydrate, protein, amino acids, osmotic pressure controlling agents,carrier gases, pH-controlling agents, organic solvents, hydrophobicagents, enzyme inhibitors, water absorbing polymers, surfactants,absorption promoters and anti-oxidative agents. Representative examplesof carbohydrates include soluble sugars such as hydropropyl cellulose,carboxymethyl cellulose, sodium carboxyl cellulose, hyaluronic acid,chitosan, alginate, glucose, xylose, galactose, fructose, maltose,saccharose, dextran, chondroitin sulfate, etc. Representative examplesof proteins include albumin, gelatin, etc. Representative examples ofamino acids include glycine, alanine, glutamic acid, arginine, lysine,and their salts.

In some embodiments, the immunogens as described herein can besolubilized in water, a solvent such as methanol, or a buffer. Suitablebuffers include, but are not limited to, phosphate buffered salineCa²⁺/Mg²⁺ free (PBS), normal saline (150 mM NaCl in water), and Trisbuffer. Antigen not soluble in neutral buffer can be solubilized in 10mM acetic acid and then diluted to the desired volume with a neutralbuffer such as PBS. In the case of antigen soluble only at acid pH,acetate-PBS at acid pH may be used as a diluent after solubilization indilute acetic acid. Glycerol can be a suitable non-aqueous buffer foruse in the present invention.

If the immunogen as disclosed herein is not soluble per se, theimmunogen can be present in the formulation in a suspension or even asan aggregate. In some embodiments, hydrophobic antigen can besolubilized in a detergent, for example a polypeptide containing amembrane-spanning domain. Furthermore, for formulations containingliposomes, an antigen in a detergent solution (e.g., a cell membraneextract) may be mixed with lipids, and liposomes then may be formed byremoval of the detergent by dilution, dialysis, or columnchromatography.

In some embodiments, a vaccine composition is administered incombination with other therapeutic ingredients including, e.g.,γ-interferon, cytokines, chemotherapeutic agents, or anti-inflammatoryor anti-viral agents.

In some embodiments, a vaccine composition is administered in a pure orsubstantially pure form, but it is preferable to present it as apharmaceutical composition, formulation or preparation. Such formulationcomprises polypeptides described herein together with one or morepharmaceutically acceptable carriers and optionally other therapeuticingredients. Other therapeutic ingredients include compounds thatenhance antigen presentation, e.g., gamma interferon, cytokines,chemotherapeutic agents, or anti-inflammatory agents. The formulationscan conveniently be presented in unit dosage form and may be prepared bymethods well known in the pharmaceutical art. For example, Plotkin andMortimer (In ‘Vaccines’, 1994, W.B. Saunders Company; 2nd edition)describes vaccination of animals or humans to induce an immune responsespecific for particular pathogens, as well as methods of preparingantigen, determining a suitable dose of antigen, and assaying forinduction of an immune response.

In some embodiments, a vaccine composition as described herein furthercomprises an adjuvant, as described herein.

Formulations of vaccine compositions suitable for intravenous,intramuscular, intranasal, oral, subcutaneous, or intraperitonealadministration conveniently comprise sterile aqueous solutions of theactive ingredient with solutions which are preferably isotonic with theblood of the recipient. Such formulations may be conveniently preparedby dissolving solid active ingredient in water containingphysiologically compatible substances such as sodium chloride (e.g.,0.1-2.0 M), glycine, and the like, and having a buffered pH compatiblewith physiological conditions to produce an aqueous solution, andrendering the solution sterile. These may be present in unit ormulti-dose containers, for example, sealed ampoules or vials.

Liposomal suspensions can also be used as pharmaceutically acceptablecarriers. These can be prepared according to methods known to thoseskilled in the art, for example, as described in U.S. Pat. No.4,522,811.

Formulations for an intranasal delivery are described in U.S. Pat. Nos.5,427,782, 5,843,451 and 6,398,774, which are incorporated herein intheir entirety by reference. Other means of mucosal administration arealso encompassed herein.

The formulations of a vaccine composition as disclosed herein can alsoincorporate a stabilizer. Illustrative stabilizers are polyethyleneglycol, proteins, saccharide, amino acids, inorganic acids, and organicacids which may be used either on their own or as admixtures. Two ormore stabilizers may be used in aqueous solutions at the appropriateconcentration and/or pH. The specific osmotic pressure in such aqueoussolution is generally in the range of 0.1-3.0 osmoses, preferably in therange of 0.80-1.2. The pH of the aqueous solution is adjusted to bewithin the range of 5.0-9.0, preferably within the range of 6-8.

When oral preparations are desired, a vaccine composition can becombined with typical carriers, such as lactose, sucrose, starch, talcmagnesium stearate, crystalline cellulose, methyl cellulose,carboxymethyl cellulose, glycerin, sodium alginate or gum arabic amongothers.

The present invention provides immunogenic compositions (e.g., vaccines)comprising a novel pneumococcal antigen, e.g., one or more of apolypeptide antigen selected from Table 1 or functional fragmentsthereof and any combinations thereof, and one or more pharmaceuticallyacceptable excipients. In accordance with some embodiments, a method ofadministering an inventive immunogenic composition to a subject in needthereof is provided. In some embodiments, inventive compositions areadministered to humans. For the purposes of the present invention, thephrase “active ingredient” generally refers to an inventive immunogeniccomposition comprising at least one chlamydia antigen and optionallycomprising one or more additional agents, such as an adjuvant.

Although the descriptions of immunogenic compositions provided hereinare principally directed to compositions which are suitable foradministration to humans, it will be understood by the skilled artisanthat such compositions are generally suitable for administration toanimals of all sorts. Modification of immunogenic compositions suitablefor administration to humans in order to render the compositionssuitable for administration to various animals is well understood, andthe ordinarily skilled veterinary pharmacologist can design and/orperform such modification with merely ordinary, if any, experimentation.Subjects to which administration of the immunogenic compositions of theinvention is contemplated include, but are not limited to, humans and/orother primates; mammals, including commercially relevant mammals such asdomestic animals, cattle, pigs, horses, sheep, cats, and/or dogs; and/orbirds, including commercially relevant birds such as chickens, ducks,geese, and/or turkeys.

The formulations of the immunogenic compositions described herein may beprepared by any method known or hereafter developed in the art ofvaccines. In some embodiments, such preparatory methods include the stepof bringing the antigen(s) (or nucleic acids encoding the antigens, fornucleic acid based applications) into association with one or moreexcipients and/or one or more other accessory ingredients, and then, ifnecessary and/or desirable, shaping and/or packaging the product into adesired single- or multi-dose unit.

An immunogenic composition of the invention may be prepared, packaged,and/or sold in bulk, as a single unit dose, and/or as a plurality ofsingle unit doses. As used herein, a “unit dose” is discrete amount ofthe immunogenic composition comprising a predetermined amount of theantigen(s).

The relative amounts of the antigen(s), the pharmaceutically acceptableexcipient(s), and/or any additional ingredients (e.g., adjuvant) in acomposition of the invention will vary, depending upon the identity,size, and/or condition of the subject treated and further depending uponthe route by which the composition is to be administered.

Immunogenic formulations of the present invention may additionallycomprise a pharmaceutically acceptable excipient, which, as used herein,includes any and all solvents, dispersion media, diluents, or otherliquid vehicles, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, solidbinders, lubricants and the like, as suited to the particular dosageform desired. Remington's The Science and Practice of Pharmacy,21.sup.st Edition, A. R. Gennaro, (Lippincott, Williams & Wilkins,Baltimore, Md., 2006; incorporated herein by reference) disclosesvarious excipients used in formulating pharmaceutical compositions andknown techniques for the preparation thereof. Except insofar as anyconventional excipient is incompatible with a substance or itsderivatives, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the immunogenic composition, its use is contemplated tobe within the scope of this invention.

In some embodiments, the pharmaceutically acceptable excipient is atleast 95%, 96%, 97%, 98%, 99%, or 100% pure. In some embodiments, theexcipient is approved for use in humans and for veterinary use. In someembodiments, the excipient is approved by United States Food and DrugAdministration. In some embodiments, the excipient is pharmaceuticalgrade. In some embodiments, the excipient meets the standards of theUnited States Pharmacopoeia (USP), the European Pharmacopoeia (EP), theBritish Pharmacopoeia, and/or the International Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture ofimmunogenic compositions include, but are not limited to, inertdiluents, dispersing and/or granulating agents, surface active agentsand/or emulsifiers, disintegrating agents, binding agents,preservatives, buffering agents, lubricating agents, and/or oils. Suchexcipients may optionally be included in the inventive formulations.

Injectable formulations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Asterile injectable preparation may be a sterile injectable solution,suspension or emulsion in a nontoxic parenterally acceptable diluent orsolvent, for example, as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedor dispersed in sterile water or other sterile injectable medium priorto use.

In order to prolong release of an immunogenic composition and stimulatemaximal uptake by antigen presenting cells in the vicinity of aninjection site, it is often desirable to slow the absorption fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. Alternatively, delayed absorption of aparenterally administered drug form may be accomplished by dissolving orsuspending the drug in an oil vehicle.

In some embodiments, an immunogenic composition is administered to amucosal surface. Compositions for rectal or vaginal administration caninclude suppositories which can be prepared by mixing immunogeniccompositions of this invention with suitable excipients such as cocoabutter, polyethylene glycol or a suppository wax, which are solid atambient temperature but liquid at body temperature and therefore melt inthe rectum or vaginal cavity and release antigen.

In some embodiments, an immunogenic composition is administered orally.Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the antigencan be mixed with at least one inert, pharmaceutically acceptableexcipient such as sodium citrate or dicalcium phosphate and/or a)fillers or extenders such as starches, lactose, sucrose, glucose,mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may comprise buffering agents.

Suitable devices for use in delivering immunogenic compositions by anintradermal route described herein include short needle devices such asthose described in U.S. Pat. Nos. 4,886,499; 5,190,521; 5,328,483;5,527,288; 4,270,537; 5,015,235; 5,141,496; and 5,417,662. Jet injectiondevices which deliver liquid immunogenic compositions to the dermis viaa liquid jet injector and/or via a needle which pierces the stratumcorneum and produces a jet which reaches the dermis are suitable. Jetinjection devices are described, for example, in U.S. Pat. Nos.5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189;5,704,911; 5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335;5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880;4,940,460; and PCT publications WO 97/37705 and WO 97/13537. Ballisticpowder/particle delivery devices which use compressed gas to acceleratean immunogenic composition in powder form through the outer layers ofthe skin to the dermis are suitable. Alternatively or additionally,conventional syringes may be used in the classical mantoux method ofintradermal administration.

General considerations in the formulation and/or manufacture ofpharmaceutical agents may be found, for example, in Remington: TheScience and Practice of Pharmacy 21.sup.st ed., Lippincott Williams &Wilkins, 2005.

The vaccine formulation or immunogenic composition may be suitable foradministration to a human patient, and vaccine or immunogeniccomposition preparation may conform to USFDA guidelines. In someembodiments, the vaccine formulation or immunogenic composition issuitable for administration to a non-human animal. In some embodiments,the vaccine or immunogenic composition is substantially free of eitherendotoxins or exotoxins. Endotoxins may include pyrogens, such aslipopolysaccharide (LPS) molecules. The vaccine or immunogeniccomposition may also be substantially free of inactive protein fragmentswhich may cause a fever or other side effects. In some embodiments, thecomposition contains less than 1%, less than 0.1%, less than 0.01%, lessthan 0.001%, or less than 0.0001% of endotoxins, exotoxins, and/orinactive protein fragments. In some embodiments, the vaccine orimmunogenic composition has lower levels of pyrogens than industrialwater, tap water, or distilled water. Other vaccine or immunogeniccomposition components may be purified using methods known in the art,such as ion-exchange chromatography, ultrafiltration, or distillation.In other embodiments, the pyrogens may be inactivated or destroyed priorto administration to a patient. Raw materials for vaccines, such aswater, buffers, salts and other chemicals may also be screened anddepyrogenated. All materials in the vaccine may be sterile, and each lotof the vaccine may be tested for sterility. Thus, in certain embodimentsthe endotoxin levels in the vaccine fall below the levels set by theUSFDA, for example 0.2 endotoxin (EU)/kg of product for an intrathecalinjectable composition; 5 EU/kg of product for a non-intrathecalinjectable composition, and 0.25-0.5 EU/mL for sterile water.

In certain embodiments, the preparation comprises less than 50%, 20%,10%, or 5% (by dry weight) contaminating protein. In certainembodiments, the desired molecule is present in the substantial absenceof other biological macromolecules, such as other proteins (particularlyother proteins which may substantially mask, diminish, confuse or alterthe characteristics of the component proteins either as purifiedpreparations or in their function in the subject reconstituted mixture).In certain embodiments, at least 80%, 90%, 95%, 99%, or 99.8% (by dryweight) of biological macromolecules of the same type present (butwater, buffers, and other small molecules, especially molecules having amolecular weight of less than 5000, can be present). In someembodiments, the vaccine or immunogenic composition comprising purifiedsubunit proteins contains less than 5%, 2%, 1%, 0.5%, 0.2%, 0.1% ofprotein from host cells in which the subunit proteins were expressed,relative to the amount of purified subunit. In some embodiments, thedesired polypeptides are substantially free of nucleic acids and/orcarbohydrates. For instance, in some embodiments, the vaccine orimmunogenic composition contains less than 5%, less than 2%, less than1%, less than 0.5%, less than 0.2%, or less than 0.1% host cell DNAand/or RNA. In certain embodiments, at least 80%, 90%, 95%, 99%, or99.8% (by dry weight) of biological macromolecules of the same type arepresent in the preparation (but water, buffers, and other smallmolecules, especially molecules having a molecular weight of less than5000, can be present).

It is preferred that the vaccine or immunogenic composition has low orno toxicity, within a reasonable risk-benefit ratio. In certainembodiments, the vaccine or immunogenic composition comprisesingredients at concentrations that are less than LD.50 measurements forthe animal being vaccinated. LD50 measurements may be obtained in miceor other experimental model systems, and extrapolated to humans andother animals. Methods for estimating the LD50 of compounds in humansand other animals are well-known in the art. A vaccine formulation orimmunogenic composition, and any component within it, might have an LD50value in rats of greater than 100 g/kg, greater than 50 g/kg, greaterthan 20 g/kg, greater than 10 g/kg, greater than 5 g/kg, greater than 2g/kg, greater than 1 g/kg, greater than 500 mg/kg, greater than 200mg/kg, greater than 100 mg/kg, greater than 50 mg/kg, greater than 20mg/kg, or greater than 10 mg/kg. A vaccine formulation or immunogeniccomposition that comprises a toxin such as botulinum toxin (which can beused as an adjuvant) should contain significantly less than the LD50 ofbotulinum toxin.

The formulations suitable for introduction of the vaccine formulationsor pharmaceutical composition vary according to route of administration.Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, intranasal, and subcutaneous routes,include aqueous and non-aqueous, isotonic sterile injection solutions,which can contain antioxidants, buffers, bacteriostats, and solutes thatrender the formulation isotonic with the blood of the intendedrecipient, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. The formulations can be presented in unit-dose ormulti-dose sealed containers, such as ampoules and vials.

Injection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described. In thecase of adoptive transfer of therapeutic T cells, the cells can beadministered intravenously or parenterally.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the polypeptides or packagednucleic acids suspended in diluents, such as water, saline or PEG 400;(b) capsules, sachets or tablets, each containing a predetermined amountof the active ingredient, as liquids, solids, granules or gelatin; (c)suspensions in an appropriate liquid; and (d) suitable emulsions. Tabletforms can include one or more of lactose, sucrose, mannitol, sorbitol,calcium phosphates, corn starch, potato starch, tragacanth,microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide,croscarmellose sodium, talc, magnesium stearate, stearic acid, and otherexcipients, colorants, fillers, binders, diluents, buffering agents,moistening agents, preservatives, flavoring agents, dyes, disintegratingagents, and pharmaceutically compatible carriers. Lozenge forms cancomprise the active ingredient in a flavor, usually sucrose and acaciaor tragacanth, as well as pastilles comprising the active ingredient inan inert base, such as gelatin and glycerin or sucrose and acaciaemulsions, gels, and the like containing, in addition to the activeingredient, carriers known in the art. The pharmaceutical compositionscan be encapsulated, e.g., in liposomes, or in a formulation thatprovides for slow release of the active ingredient.

The antigens, alone or in combination with other suitable components,can be made into aerosol formulations (e.g., they can be “nebulized”) tobe administered via inhalation. Aerosol formulations can be placed intopressurized acceptable propellants, such as dichlorodifluoromethane,propane, nitrogen, and the like. Aerosol formulations can be deliveredorally or nasally.

Suitable formulations for vaginal or rectal administration include, forexample, suppositories, which consist of the polypeptides or packagednucleic acids with a suppository base. Suitable suppository basesinclude natural or synthetic triglycerides or paraffin hydrocarbons. Inaddition, it is also possible to use gelatin rectal capsules whichconsist of a combination of the polypeptides or packaged nucleic acidswith a base, including, for example, liquid triglycerides, polyethyleneglycols, and paraffin hydrocarbons.

Doses and Routes of Administration

1. Dosage Forms, Amounts, and Timing

The amount of antigen in each vaccine or immunogenic composition dose isselected as an effective amount, which induces a prophylactic ortherapeutic response, as described above, in either a single dose orover multiple doses. Preferably, the dose is without significant adverseside effects in typical vaccines. Such amount will vary depending uponwhich specific antigen is employed. Generally, it is expected that adose will comprise 1-1000 μg of each protein, in some instances 2-100μg, for instance 4-40 μg. In some aspects, the vaccine formulationcomprises 1-1000 μg of the polypeptide and 1-250 μg of the adjuvant. Insome embodiments, the appropriate amount of antigen to be delivered willdepend on the age, weight, and health (e g immunocompromised status) ofa subject. When present, typically an adjuvant will be present inamounts from 1 μg-250 μg per dose, for example 50-150 μg, 75-125 μg or100 μg.

In some embodiments, only one dose of the vaccine is administered toachieve the results described above. In other embodiments, following aninitial vaccination, subjects receive one or more boost vaccinations,for a total of two, three, four or five vaccinations. Advantageously,the number is three or fewer. A boost vaccination may be administered,for example, about 1 month, 2 months, 4 months, 6 months, or 12 monthsafter the initial vaccination, such that one vaccination regimeninvolves administration at 0, 0.5-2 and 4-8 months. It may beadvantageous to administer split doses of vaccines which may beadministered by the same or different routes.

The vaccines and immunogenic compositions described herein may take on avariety of dosage forms. In certain embodiments, the composition isprovided in solid or powdered (e.g., lyophilized) form; it also may beprovided in solution form. In certain embodiments, a dosage form isprovided as a dose of lyophilized composition and at least one separatesterile container of diluent.

In some embodiments, the composition will be administered in a doseescalation manner, such that successive administrations of thecomposition contain a higher concentration of composition than previousadministrations. In some embodiments, the composition will beadministered in a manner such that successive administrations of thecomposition contain a lower concentration of composition than previousadministrations.

The precise dose to be employed in the formulation will also depend onthe route of administration and should be decided according to thejudgment of the practitioner and each patient's circumstances. Forexample, a range of 25 μg-900 μg total immunogen protein can beadministered intradermally, monthly for 3 months. Ultimately, theattending physician will decide the amount of protein or vaccinecomposition to administer to particular individuals.

In some embodiments, a therapeutically effective amount of an inventiveimmunogenic composition is delivered to a patient and/or animal priorto, simultaneously with, and/or after exposure to a chlamydia organismor diagnosis with a chlamydial disease, disorder, and/or condition. Insome embodiments, a therapeutic amount of an inventive composition isdelivered to a patient and/or animal prior to, simultaneously with,and/or after onset of symptoms of a chlamydial disease, disorder, and/orcondition. In some embodiments, the amount of an immunogenic compositionis sufficient to reduce risk of infection by, or treat, alleviate,ameliorate, relieve, delay onset of, inhibit progression of, reduceseverity of, and/or reduce incidence of one or more symptoms or featuresof the chlamydial disease, disorder, and/or condition.

Immunogenic compositions, according to the method of the presentinvention, may be administered using any amount and any route ofadministration effective for treatment. The exact amount required willvary from subject to subject, depending on the species, age, and generalcondition of the subject, the severity of the infection, the particularcomposition, its mode of administration, its mode of activity, and thelike. The specific effective dose level for any particular subject ororganism will depend upon a variety of factors including theimmunogenicity of the antigen composition employed; the specificcomposition employed; the nature of adjuvant used; the age, body weight,general health, sex and diet of the subject; the time of administration,route of administration, and like factors well known in the medicalarts.

In therapeutic applications, compositions are administered to a patientsuffering from a disease in an amount sufficient to treat the patient.Therapeutic applications of a composition described herein includereducing transmissibility, slowing disease progression, reducingbacterial viability or replication, or inhibiting the expression ofproteins required for toxicity, such as by 90%, 80%, 70%, 60%, 50%, 40%,30%, 20% or 10% of the levels at which they would occur in individualswho are not treated with the composition.

In prophylactic embodiments, compositions are administered to a human orother mammal to induce an immune response that can inhibit theestablishment of an infectious disease or other condition. In someembodiments, a composition may partially block the bacterium fromestablishing an infection.

In some embodiments, the compositions are administered in combinationwith antibiotics. This co-administration is particularly appropriatewhen the pharmaceutical composition is administered to a patient who hasrecently been exposed (or is suspected of having been recently exposed)to S. pneumoniae. Many antibiotics are used to treat pneumococcalinfections, including penicillin, amoxicillin, amoxicillin/clavulanate,cefuroxime, cefotaxime, ceftriaxone, and vancomycin. The appropriateantibiotic may be selected based on the type and severity of theinfection, as well as any known antibiotic resistance of the infection(Jacobs M R “Drug-resistant Streptococcus pneumoniae: rationalantibiotic choices” Am J. Med. 1999 May 3; 106 (5A):19S-25S).

2. Administration

A method of immunization or vaccinating a mammal against pneumococcalinfections comprises administering a vaccine composition describedherein.

In some embodiments, the immunogenic compositions or vaccinecompositions as described herein can be administered intravenously,intranasally, intramuscularly, subcutaneously, infraperitoneally ororally. A preferred route of administration is intranasal or by othermucosal route.

Vaccination can be conducted by conventional methods. For example, animmunogen as polypeptide as disclosed in Table 1 can be used in asuitable diluent such as saline or water, and optionally with completeor incomplete adjuvants. The vaccine can be administered by any routeappropriate for eliciting an immune response. The vaccine can beadministered once or at periodic intervals until an immune response iselicited Immune responses can be detected by a variety of methods knownto those skilled in the art, including but not limited to, antibodyproduction, cytotoxicity assay, proliferation assay and cytokine releaseassays. For example, samples of blood can be drawn from the immunizedmammal, and analyzed for the presence of antibodies against theimmunogen protein used in the vaccination by ELISA and the titer ofthese antibodies can be determined by methods known in the art.

Immunogenic compositions of the present invention may be administered byany route that elicits an immune response. In some embodiments, animmunogenic composition is administered subcutaneously. In someembodiments, an immunogenic composition is administered intramuscularly.In some embodiments, the immunogenic compositions of the presentinvention are administered by a variety of routes, including oral,intravenous, intra-arterial, intramedullary, intrathecal,intraventricular, transdermal, interdermal, rectal, intravaginal,intraperitoneal, topical (as by powders, ointments, creams, and/ordrops), transdermal, mucosal, nasal, buccal, enteral, sublingual; byintratracheal instillation, bronchial instillation, and/or inhalation;and/or as an oral spray, nasal spray, and/or aerosol.

The vaccine formulations and pharmaceutical compositions herein can bedelivered by administration to an individual, typically by systemicadministration (e.g., intravenous, intraperitoneal, intramuscular,intradermal, subcutaneous, subdermal, transdermal, intracranial,intranasal, mucosal, anal, vaginal, oral, buccal route or they can beinhaled) or they can be administered by topical application. In someembodiments, the route of administration is intramuscular. In otherembodiments, the route of administration is subcutaneous. In yet otherembodiments, the route of administration is mucosal. In certainembodiments, the route of administration is transdermal or intradermal

Certain routes of administration are particularly appropriate forvaccine formulations and immunogenic compositions comprising specifiedadjuvants. In particular, transdermal administration is one suitableroute of administration for S. pneumoniae vaccines comprising toxins(e.g. cholera toxin or labile toxin); in other embodiments, theadministration is intranasal. Vaccines formulated with Alphavirusreplicons may be administered, for example, by the intramuscular or thesubcutaneous route. Vaccines comprising Monophosphory Lipid A (MPL),Trehalose Dicoynomycolate (TDM), and dioctadecyldimethylammonium bromide(DDA) are suitable (inter alia) for intramuscular and subcutaneousadministration. A vaccine comprising resiquimod may be administeredtopically or subcutaneously, for example.

In certain embodiments, an immunogenic composition of the invention maybe administered in amounts that include a protein antigen in ranges of 1μg-500 μg. In some embodiments, a dose of about 10 μg, 20, 30 μg, 50 μg,or 100 μg is administered to a human.

In some embodiments, an immunogenic composition is administered morethan once (e.g., twice, three times, four times, five times). In someembodiments, a boost is given about one week, two weeks, three weeks,one month, three months, six months, one year, or longer after aninitial immunization.

Preparation and Storage of Vaccine Formulations and ImmunogenicCompositions

The S. pneumoniae vaccines and immunogenic compositions described hereinmay be produced using a variety of techniques. For example, apolypeptide may be produced using recombinant DNA technology in asuitable host cell. A suitable host cell may be bacterial, yeast,mammalian, or other type of cell. The host cell may be modified toexpress an exogenous copy of one of the relevant polypeptide genes.Typically, the gene is operably linked to appropriate regulatorysequences such as a strong promoter and a polyadenylation sequence. Insome embodiments, the promoter is inducible or repressible. Otherregulatory sequences may provide for secretion or excretion of thepolypeptide of interest or retention of the polypeptide of interest inthe cytoplasm or in the membrane, depending on how one wishes to purifythe polypeptide. The gene may be present on an extrachromosomal plasmid,or may be integrated into the host genome. One of skill in the art willrecognize that it is not necessary to use a nucleic acid 100% identicalto the naturally-occurring sequence. Rather, some alterations to thesesequences are tolerated and may be desirable. For instance, the nucleicacid may be altered to take advantage of the degeneracy of the geneticcode such that the encoded polypeptide remains the same. In someembodiments, the gene is codon-optimized to improve expression in aparticular host. The nucleic acid may be produced, for example, by PCRor by chemical synthesis.

Once a recombinant cell line has been produced, a polypeptide may beisolated from it. The isolation may be accomplished, for example, byaffinity purification techniques or by physical separation techniques(e.g., a size column).

In a further aspect of the present disclosure, there is provided amethod of manufacture comprising mixing one or more polypeptides or animmunogenic fragment or variant thereof with a carrier and/or anadjuvant.

In some embodiments, antigens for inclusion the vaccine formulations andimmunogenic compositions may be produced in cell culture. One methodcomprises providing one or more expression vectors and cloningnucleotides encoding one or more polypeptides selected from polypeptideshaving an amino acid sequence of Table 1, such as SEQ ID NO: 1-76 or153-234, then expressing and isolating the polypeptides.

The immunogenic polypeptides described herein, and nucleic acidcompositions that express the polypeptides, can be packaged in packs,dispenser devices, and kits for administering nucleic acid compositionsto a mammal. For example, packs or dispenser devices that contain one ormore unit dosage forms are provided. Typically, instructions foradministration of the compounds will be provided with the packaging,along with a suitable indication on the label that the compound issuitable for treatment of an indicated condition, such as thosedisclosed herein.

Use of Immunogenic Compositions

1. Defense Against S. pneumoniae Infection

The immunogenic compositions of the present disclosure are designed toelicit an immune response against S. pneumoniae. Compositions describedherein (e.g., ones comprising one or more polypeptides of Table 1 or 2,or nucleic acids encoding the polypeptides) may stimulate an antibodyresponse or a cell-mediated immune response, or both, in the mammal towhich it is administered. In some embodiments, the compositionstimulates a TH1-biased CD4+ T cell response, a TH17-biased CD4+ T cellresponse and/or a CD8+ T cell response. In some embodiments, thecomposition stimulates an antibody response. In some embodiments, thecomposition stimulates a TH1-biased CD4+ T cell response, TH17-biasedCD4+ T cell response and/or a CD8+ T cell response, and an antibodyresponse.

In certain embodiments, the composition (e.g., one comprising one ormore polypeptides of Table 1 or 2, or nucleic acids encoding thepolypeptides, or antibodies reactive with the peptides) includes acytokine or nucleotide coding region encoding a cytokine such as IL-17,to provide additional stimulation to the immune system of the mammal. Incertain embodiments, the composition comprises a cytokine such as IL-17.

While not wishing to be bound by theory, in some embodiments a T.sub.H17cell response is desirable in mounting an immune response to thecompositions disclosed herein, e.g., ones comprising one or morepolypeptides of Table 1 or 2. In certain embodiments, an activeT.sub.H17 response is beneficial in clearing a pneumococcal infection.For instance, mice lacking the IL-17A receptor show decreased whole cellvaccine-based protection from a pneumococcal challenge (Lu et al.,“Interleukin-17A mediates acquired immunity to pneumococcalcolonization.” PLoS Pathog. 2008 Sep. 19; 4 (9)).

Thus, herein is provided a method of increasing IL-17 production byadministering the compositions described herein (e.g., ones comprisingone or more polypeptides of Table 1 or 2) to a subject. Furthermore,this application provides a method of activating T.sub.H17 cells byadministering said compositions to a subject. In certain embodiments,increased IL-17A levels result in increased pneumococcal killing byneutrophils or neutrophil-like cells, for instance by inducingrecruitment and activation of neutrophils of neutrophil-like cells. Incertain embodiments, this pneumococcal killing is independent ofantibodies and complement. However, specific antibody production andcomplement activation may be useful additional mechanisms thatcontribute to clearing of a pneumococcal infection.

Immunogenic compositions containing immunogenic polypeptides orpolynucleotides encoding immunogenic polypeptides together with apharmaceutical carrier are also provided.

In some instances, the immunogenic composition comprises one or morenucleic acids encoding one or more polypeptides of SEQ ID NOS: 1-76,such as one or more nucleic acids selected from SEQ ID Nos. 77-152. Insome embodiments these nucleic acids are expressed in the immunizedindividual, producing the encoded S. pneumoniae antigens, and the S.pneumoniae antigens so produced can produce an immunostimulatory effectin the immunized individual.

Such a nucleic acid-containing immunostimulatory composition maycomprise, for example, an origin of replication, and a promoter thatdrives expression of one or more nucleic acids encoding one or morepolypeptides of SEQ ID NOS: 1-76. Such a composition may also comprise abacterial plasmid vector into which is inserted a promoter (sometimes astrong viral promoter), one or more nucleic acids encoding one or morepolypeptides of SEQ ID NOS: 1-76, and a polyadenylation/transcriptionaltermination sequence. In some instances, the nucleic acid is DNA.

Diagnostic Uses

This application provides, inter alia, a rapid, inexpensive, sensitive,and specific method for detection of S. pneumoniae in patients. In thisrespect it should be useful to all hospitals and physicians examiningand treating patients with or at risk for S. pneumoniae infection.Detection kits can be simple enough to be set up in any local hospitallaboratory, and the antibodies and antigen-binding portions thereof canreadily be made available to all hospitals treating patients with or atrisk for S. pneumoniae infection. As used herein, “patient” refers to anindividual (such as a human) that either has an S. pneumoniae infectionor has the potential to contract an S. pneumoniae infection. A patientmay be an individual (such as a human) that has an S. pneumoniaeinfection, has the potential to contract an S. pneumoniae infection, whohas recovered from S. pneumoniae infection, and/or an individual whoseinfection status is unknown.

In some embodiments, one may perform a diagnostic assay using two ormore antibodies, each of which binds one of the antigens of Table 1detect S. pneumoniae in an individual. As disclosed in Example 5, serafrom a mouse immunized with SP0785 detected the presence to type 4 TIGR4S. pneumoniae strain. Accordingly, in some embodiment, one of theantigens for use in a diagnostic is any pneumococcal antigen selectedfrom SEQ ID NO: 1-76. The instant disclosure also provides a method ofphenotyping biological samples from patients suspected of having a S.pneumoniae infection: (a) obtaining a biological sample from a patient;(b) contacting the sample with two or more S. pneumoniae-specificantibodies or antigen-binding portions thereof under conditions thatallow for binding of the antibody or antigen-binding portion to anepitope of S. pneumoniae; where binding indicates the presence of S.pneumoniae in the sample. In some embodiments, the binding to thebiological sample is compared to binding of the same antibody to anegative control tissue, wherein if the biological sample shows thepresence of S. pneumoniae as compared to the negative control tissue,the patient is identified as likely having a S. pneumoniae infection. Insome cases, binding of one antibody indicates the presence of S.pneumoniae; in other cases, the binding of two or more antibodiesindicates the presence of S. pneumoniae. The aforementioned test may beappropriately adjusted to detect other bacterial infections, forinstance by using an antibody immunoreactive a homolog (from anotherbacterial species) of one of the proteins described in Table 1. In someembodiments, the antibodies raised against a S. pneumoniae protein inTable 1 will also bind the homolog in another Streptococcus species,especially if the homologs have a high percentage sequence identity.

Alternatively, one may use an antigen of Table 1 (such as SEQ ID NO:1-76) to detect anti-S. pneumoniae antibodies in an individual. Theinstant disclosure also provides a method of phenotyping biologicalsamples from patients suspected of having a S. pneumoniae infection: (a)obtaining a biological sample from a patient; (b) contacting the samplewith two or more S. pneumoniae-specific antigens selected from Table 1or portions or functional fragments thereof under conditions that allowfor binding of the antigen (or portion thereof) to any host antibodiespresent in the sample; where binding indicates the presence of anti-S.pneumoniae antibodies in the sample. In some embodiments, the binding tothe biological sample is compared to binding of the same antigen to anegative control tissue, wherein if the biological sample shows thepresence of anti-S. pneumoniae antibodies as compared to the negativecontrol tissue, the patient is identified as likely either (1) having aS. pneumoniae infection, or (2) having had a S. pneumoniae infection inthe past. In some cases, detecting one antibody indicates a current orpast infection with S. pneumoniae; in other cases, detecting two or moreantibodies indicates a current or past infection with S. pneumoniae. Theaforementioned test may be appropriately adjusted to detect otherbacterial infections, for instance by using a homolog (from anotherbacterial species (e.g., a Streptococcal species) of the proteinsdescribed in Table 1.

In some embodiments, the immune cell response of a mammalian cell may bequantified ex vivo. A method for such quantification comprisesadministering the compositions herein disclosed to a mammalian T cell exvivo, and quantifying the change in cytokine production of the mammalianT cell in response to the composition. In these methods, the cytokinemay be, for example, IL-17.

The binding of an S. pneumoniae antibody to an antigen (e.g., apolypeptide of Table 1, such as SEQ ID NO: 1-76) may be measured usingany appropriate method. Such methods include ELISA (enzyme-linkedimmunosorbent assay), Western blotting, competition assay, andspot-blot. The detection step may be, for instance, chemiluminescent,fluorescent, or colorimetric. One suitable method for measuringantibody-protein binding is the Luminex xMAP system, where peptides arebound to a dye-containing microsphere. Certain systems, including thexMAP system, are amenable to measuring several different markers inmultiplex, and could be used to measure levels of antibodies at once. Insome embodiments, other systems are used to assay a plurality of markersin multiplex. For example, profiling may be performed using any of thefollowing systems: antigen microarrays, bead microarrays, nanobarcodesparticle technology, arrayed proteins from cDNA expression libraries,protein in situ array, protein arrays of living transformants, universalprotein array, lab-on-a-chip microfluidics, and peptides on pins.Another type of clinical assay is a chemiluminescent assay to detectantibody binding. In some such assays, including the VITROS Eci anti-HCVassay, antibodies are bound to a solid-phase support made up ofmicroparticles in liquid suspension, and a surface fluorometer is usedto quantify the enzymatic generation of a fluorescent product.

In some embodiments, if the biological sample shows the presence of S.pneumoniae (e.g., by detecting one or more polypeptide of Table 1, suchas SEQ ID NO: 1-76, or an antibody that binds one of said polypeptides),one may administer a therapeutically effective amount of thecompositions and therapies described herein to the patient. Thebiological sample may comprise, for example, blood, semen, urine,vaginal fluid, mucus, saliva, feces, urine, cerebrospinal fluid, or atissue sample. In some embodiments, the biological sample is an organintended for transplantation. In certain embodiments, before thedetection step, the biological sample is subject to culture conditionsthat promote the growth of S. pneumoniae.

The diagnostic tests herein (e.g., those that detect a polypeptide ofTable 1, such as SEQ ID NO: 1-76, or an antibody that binds one of saidpolypeptides) may be used to detect S. pneumoniae in a variety ofsamples, including samples taken from patients and samples obtained fromother sources. For example, the diagnostic tests may be used to detectS. pneumoniae in food, drink, or ingredients for food and drink; onobjects such as medical instruments, medical devices such as cochlearimplants and pacemakers, shoes, clothing, furniture including hospitalfurniture, and drapes including hospital drapes; or in samples takenfrom the environment such as plant samples. In some embodiments, thetests herein may be performed on samples taken from animals such asagricultural animals (cows, pigs, chickens, goats, horses and the like),companion animals (dogs, cats, birds, and the like), or wild animals. Incertain embodiments, the tests herein may be performed on samples takenfrom cell cultures such as cultures of human cells that produce atherapeutic protein, cultures of bacteria intended to produce a usefulbiological molecule, or cultures of cells grown for research purposes.

This disclosure also provides a method of determining the location of aS. pneumoniae infection in a patient comprising: (a) administering apharmaceutical composition comprising a labeled S. pneumoniae antibodyor antigen-binding portion thereof to the patient, and (b) detecting thelabel, wherein binding indicates a S. pneumoniae infection in aparticular location in the patient. Such a diagnostic may also comprisecomparing the levels of binding in the patient to a control. In certainembodiments, the method further comprises, if the patient has a S.pneumoniae infection, treating the infection by administering atherapeutically effective amount of a S. pneumoniae-binding antibody orantigen-binding portion thereof to the patient. In certain embodiments,the method further comprises, if the patient has a S. pneumoniaeinfection, treating the infection by administering a therapeuticallyeffective amount of a S. pneumoniae protein of Table 1 or 2, orimmunogenic portion thereof, to the patient. The method may furthercomprise determining the location and/or volume of the S. pneumoniae inthe patient. This method may be used to evaluate the spread of S.pneumoniae in the patient and determine whether a localized therapy isappropriate.

In some embodiments, the anti-S. pneumoniae antibodies or T cellsdescribed herein may be used to make a prognosis of the course ofinfection. In some embodiments, the anti-S. pneumoniae antibodies or Tcells herein may be detected in a sample taken from a patient. Ifantibodies or T cells are present at normal levels, it would indicatethat the patient has raised an immune response against anti-S.pneumoniae. If antibodies or T cells are absent, or present at reducedlevels, it would indicate that the patient is failing to raise asufficient response against anti-S. pneumoniae, and a more aggressivetreatment would be recommended. In some embodiments, antibodies or Tcells present at reduced levels refers to antibodies that are present atless than 50%, 20%, 10%, 5%, 2%, or 1% the level of antibodies or Tcells typical in a patient with a normal immune system. Antibodies maybe detected by affinity for any of the antigens described herein (e.g.,those in Table 1 and/or 2), for example using ELISA. T cells may bedetected by ex vivo responses for any of the antigens described herein(e.g., those in Table 1 and/or 2), for example using ELISA or ELISPOTassays.

In some embodiments, detection of specific S. pneumoniae antigens (e.g.,those in Table 1 and/or 2, such as SEQ ID NO: 1-76 and/or SEQ ID NO:153-234) may be used to predict the progress and symptoms of S.pneumoniae infection in a patient. It will be understood by one of skillin the art that the methods herein are not limited to detection of S.pneumoniae. Other embodiments include the detection of related bacteriaincluding bacteria with proteins homologous to the proteins described inTable 1 or 2. Such related bacteria include, for example, other strainsof Streptococcus. Accordingly, in some embodiments, such pneumococcalantigen proteins of Table 1 or immunogen mixtures may be useful indiagnostics.

Kits

Another aspect of the present invention provides a variety of kitscomprising one or more of the pneumococcal antigens as described herein.For example, the invention provides a kit including a novel pneumococcalantigen and instructions for use. A kit may include multiple differentpneumococcal antigens. A kit may include any of a number of additionalcomponents or reagents in any combination. All of the variouscombinations are not set forth explicitly but each combination isincluded in the scope of the invention.

According to certain embodiments of the invention, a kit may include,for example, (i) an immunogenic composition including at least one ofthe following chlamydia antigens: SP0785, SP1500, SP0346, SP1386,SP0084, SP1479 and SP2145 polypeptide antigens; and (ii) instructionsfor administering the composition to a subject in need thereof. In someembodiments, the kit further includes an adjuvant.

Kits that include nucleic acids encoding chlamydia antigens are alsoprovided. In certain embodiments, a kit may include, for example, (i) acomposition including a nucleic acid encoding a pneumococcal antigen;(ii) instructions for use of the nucleic acid compositing (e.g.,instructions for expressing the nucleic acid for producing the antigen,or instructions for administering the composition to a subject in needthereof to elicit a response against pneumococcus).

Instructions included with kits may, for example, include protocolsand/or describe conditions for production of immunogenic compositionsand/or administration of immunogenic compositions, to a subject in needthereof, etc. Kits generally include one or more vessels or containersso that some or all of the individual components and reagents may beseparately housed. Kits may also include a means for enclosingindividual containers in relatively close confinement for commercialsale, e.g., a plastic box, in which instructions, packaging materialssuch as styrofoam, etc., may be enclosed. An identifier, e.g., a barcode, radio frequency identification (ID) tag, etc., may be present inor on the kit or in or one or more of the vessels or containers includedin the kit. An identifier can be used, e.g., to uniquely identify thekit for purposes of quality control, inventory control, tracking,movement between workstations, etc.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   -   1. An immunogenic composition comprising at least one isolated        pneumococcal antigen or fragment thereof with the amino acid        sequence selected from SEQ ID NO: 1-76 or SEQ ID NO: 153-234,        and wherein the composition elicits an immune response against        Streptococcus pneumoniae when administered to a mammal.    -   2. The immunogenic composition of paragraph 1, wherein the        pneumococcal antigen or fragment thereof exists as a fusion        conjugate.    -   3. The immunogenic composition of paragraph 2, wherein the        fusion conjugate is a polysaccharide conjugate.    -   4. The immunogenic composition of paragraph 3, wherein the        fusion conjugate comprises the pneumococcal antigen or fragment        thereof fused to a pneumococcal pneumolysoid PdT, wherein the        pneumococcal pneumolysoid PdT is conjugated to the        polysaccharide.    -   5. The immunogenic composition of any of paragraphs 2 to 4,        wherein the polysaccharide is dextran, Vi polysaccharide of        Salmonella typhi, or pneumococcal cell wall polysaccharide        (CWPS), or another polysaccharide of prokaryotic or eukaryotic        origin.    -   6. The immunogenic composition of any of paragraphs 1 to 5,        wherein the immunogenic composition induces a IL-17A (Th17-cell)        response in a subject.    -   7. The immunogenic composition of any of paragraphs 1 to 6,        wherein the immunogenic composition is further prepared as a        vaccine that reduces or protects a mammal against pneumococcal        colonization.    -   8. The immunogenic composition of any of paragraphs 1 to 7,        wherein the immunogenic composition further comprises an        adjuvant.    -   9. The immunogenic composition of any of paragraphs 1 to 8,        wherein said immunogenic composition is administered mucosally.    -   10. The immunogenic composition of any of paragraphs 1 to 9,        wherein the immunogenic composition comprises at least 2        pneumococcal antigens or fragments with the amino acid sequence        selected from SEQ ID NO: 1-76 or SEQ ID NO: 153-234.    -   11. The immunogenic composition of any of paragraphs 1 to 10,        wherein the immunogenic composition comprises at least 3        pneumococcal antigens or fragments with the amino acid sequence        selected from SEQ ID NO: 1-76 or SEQ ID NO: 153-234.    -   12. The immunogenic composition of any of paragraphs 1 to 11,        wherein the immunogenic composition comprises at least 5        pneumococcal antigens or fragments with the amino acid sequence        selected from SEQ ID NO: 1-76 or SEQ ID NO: 153-234.    -   13. The immunogenic composition of any of paragraphs 1 to 12,        wherein the immunogenic composition comprises between 5 and 20        pneumococcal antigens or fragments thereof with the amino acid        sequence selected from SEQ ID NO: 1-76 or SEQ ID NO: 153-234.    -   14. The immunogenic composition of any of paragraphs 1 to 13,        wherein the immunogenic composition comprises more than 20        pneumococcal antigens or fragments thereof with the amino acid        sequence selected from SEQ ID NO: 1-76 or SEQ ID NO: 153-234.    -   15. The method of any of paragraphs 1 to 14, wherein a        pneumococcal protein or protein fragment is at least one of the        pneumococcal proteins SP0785, SP1500 and SP2145.    -   16. The method of any of paragraphs 1 to 15, wherein the immune        response comprises a humoral immune response.    -   17. The method of any of paragraphs 1 to 15, wherein the immune        response comprises a cellular immune response.    -   18. A method of inducing an IL-17A response in a subject,        comprising administering to the subject at least one immunogenic        composition of any of paragraphs 1-17 effective to induce an        immune response against Streptococcus pneumoniae in the subject.    -   19. A method to protect against pneumococcal colonization,        comprising administering to the subject at least one immunogenic        composition of any of paragraphs 1-17.    -   20. A method to elicit an immune response against Streptococcus        pneumoniae in a mammal, the method comprising administering to        the mammal at least one immunogenic composition comprising one        or more isolated pneumococcal antigen or fragment thereof with        the amino acid sequence selected from SEQ ID NO: 1-76 or SEQ ID        NO: 153-234 in an effective to induce an immune response against        Streptococcus pneumoniae in the subject.    -   21. A method to protect against Streptococcus pneumonia or        Salmonella typhi colonization in a mammal, comprising        administering to the mammal at least one immunogenic composition        of any of paragraphs 1-17 in effective to induce an immune        response against Streptococcus pneumoniae in the subject.    -   22. The method of any of paragraphs 18-21, wherein the        immunogenic composition comprises at least one isolated        pneumococcal antigen or fragment thereof is SP0785 or SP1500.    -   23. The method of paragraph 22, wherein the pneumococcal antigen        or fragment thereof has an amino acid sequence selected from SEQ        ID NO: 34 (SP0785) or SEQ ID NO: 51 (SP1500).    -   24. A method to protect against an invasive disease of        Streptococcus pneumoniae in a subject, comprising administering        to the subject an immunogenic composition of any of paragraphs        1-17 in effective to induce an immune response against        Streptococcus pneumoniae in the subject.    -   25. The method of paragraph 24, wherein the invasive disease is        sepsis.    -   26. The method of paragraph 24, wherein the immunogenic        composition comprises at least one isolated pneumococcal antigen        or fragment thereof is SP1386, SP1500, SP0084 and SP1479 and        SP0346.    -   27. The method of paragraph 26, wherein the pneumococcal antigen        or fragment thereof has an amino acid sequence selected from SEQ        ID NO: 46 (SP1386), SEQ ID NO: 51 (SP1500), SEQ ID NO: 4        (SP0084), SEQ ID NO: 50 (SP1479) and SEQ ID NO: 15 (SP0346).    -   28. The method of any of paragraphs 18-27, wherein the        pneumococcal antigen or fragment thereof is conjugated to the        pneumococcal pneumolysoid PdT.    -   29. The method of any of paragraphs 18-27, wherein the        pneumococcal antigen or fragment thereof is conjugated to the Vi        polysaccharide of Salmonella typhi.    -   30. The method of any of paragraphs 18-29, wherein the        pneumococcal antigen or fragment thereof is conjugated to the        pneumococcal pneumolysoid PdT and the Vi polysaccharide of        Salmonella typhi.    -   31. The method of any of paragraphs 18-30, wherein the        administration is mucosal administration.    -   32. The method of any of paragraphs 18-31, wherein the        administration is intravenous, subcutaneous or intraperitoneal        (IP) administration.    -   33. The method of any of paragraphs 18-32, wherein the immune        response comprises a humoral immune response.    -   34. The method of any of paragraphs 18-32, wherein the immune        response comprises a cellular immune response.

EXAMPLES Materials and Methods

Materials: Aluminum hydroxide (alum) was from Brenntag North America (2%Alhydrogel). Ni-NTA resin was purchased from Qiagen. CloneEZ PCR cloningkit was obtained from Genscript Inc. All other reagents were obtainedfrom Sigma or Thermo Fisher Scientific.

Construction of Expression Library.

The extracellular domains of selected proteins were amplified usingTIGR4 genomic DNA as template by PCR and then integrated into pET21bexpression vectors using the CloneEZ PCR cloning kit. Signal andtransmembrane sequences were excluded from cloning. Detailed cloningregion and primer sequences are listed in table I. For proteins that areunusually large (SP0648, SP0664 and SP1154, larger than 250 kDa) andproved difficult to purify as full-length, we divided their sequenceinto 3 parts and purified each separately. We chose to divide theproteins based on the prediction of their secondary structure byBCL::Jufo (http://meilerlab.org/index.php/servers/show?s_id=5), makingtruncations in areas that are not conserved by amino acid sequence. Thusthe final protein library consists of 80 proteins/peptides. Plasmidsequences were verified by Genewiz Inc.

Protein Purification.

E. coli transformants containing the relevant cloned proteins were grownto OD600=0.6, and protein expression was induced with 0.2 mM IPTG at 16°C. overnight. Cells were spun down and pellets were resuspended in lysisbuffer (20 mM Tris-HCl, 500 mM NaCl, pH8.0) and then lysed bysonication. The proteins of interest were purified from supernatant overa Ni-NTA column; proteins were eluted in imidazole buffer.Protein-containing elutions were combined, purified over agel-filtration column and eluted in PBS buffer.

Generation of the Vi Conjugates.

Vi was resuspended to 5 mg/ml in buffer A (0.2 M MES, 150 mM NaCl, pH5.9), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) andNHS-Sulfo was added into solution as powder for 30 minutes at 4° C.Reaction buffer was raised to pH7.5 by addition of 1M Na₂HPO₄. Proteinswere then added to the reaction as 2 mg/mg, protein/sugar. The reactionwas carried out at 4° C. for 48 hours with rotation. Conjugates wereseparated by elution through a Sephacryl S500 gel filtration column andcollection of the void volume fractions.

Stimulation of Mice Splenocytes and Human Peripheral Blood MononuclearCells (PBMC).

Spleens were harvested from either whole cell vaccine (WCV) immunizedC57BL/6 mice or mice have been repeatedly colonized with S. pneumoniaewere processed into a cellular suspension and stimulated with differentconcentration of recombinant proteins. Splenocytes were incubated for 3days; plates were then spun to pellet cells, after which thesupernatants were collected and assayed for IL-17A using a mouse IL-17AELISA kit (R&D Systems, Inc).

Human PBMC were separated from healthy adult volunteers and cultured in96 well plates. Cells were stimulated with different concentration ofrecombinant proteins for 4 days and then analyzed for IL-17A in thesupernatant using a human IL-17A ELISA kit (ebioscience).

Antigen Preparations.

For intranasal (i.n.) immunization: Vaccines were prepared the same dayof immunization by mixing purified proteins (5 μg) with 1 μg Choleratoxin (CT) per mouse in a final 20 μl of saline. For subcutaneous (s.c.)immunization: One day prior to immunization, vaccines were prepared asfollows. Frozen aliquots were thawed or lyophilized vials werereconstituted with sterile water, diluted to the appropriateconcentration, and mixed with aluminum hydroxide (alum) at the indicatedconcentration in a 15 ml conical tube, which was then tumbled overnightat 4° C. to allow for adsorption.

Immunization and Challenge of Mice.

C57BL/6J mice were used in all the experiments. The age at time of firstimmunization was between 4-6 weeks. I.n. immunization was done byinstilling 20 μl of saline, adjuvant only, or adjuvant mixed withantigen as specified atraumatically into unanesthetized mice, aprocedure that puts no immunogen into the lungs; secondary immunizationswere given after one week. S.c. immunization was performed 3 times everytwo weeks. Gently restrained, nonanesthetized mice received injectionsof 200 μl containing adjuvant with or without antigen in the lower partof the back at 2-week intervals. Blood was drawn 2 weeks after the lastimmunization, and assayed for antibody and for IL-17A production invitro upon stimulation with WCA. Nasopharyngeal colonization with theclinical pneumococcal isolate 0603 (serotype 6B) was carried out aspreviously described [4]. Intraperitoneal (IP) challenge was given 1000cfu of strain WU2-ply (an engineered strain that replaced ply withHis-tagged ply) in 200 μl PBS, and sickness or death of mice wasmonitored twice daily for 14 days. All animal studies were approved byour local animal ethics committees.

Enzyme-Linked Immunosorbent Assay (ELISA) and IL-17A Production in WholeBlood Samples.

Assays for murine antibodies for individual protein and IL-17Aproduction in whole blood were carried out as previously described [2].

Binding of Antibody to Encapsulated Tigr4 Strains.

S. pneumoniae Tigr4 strain was grown to OD600=0.8 and collected bycentrifugation. Bacteria were killed at 58° C. for 1 hour and washedwith PBS twice. Bacteria were blocked with 1 ml PBS/1% BSA rotatingovernight at 4° C. Cells were spun down and resuspended in 250 μl ofPBS-Tween/1% BSA with 1:50 dilution of heat inactivated mouse serum.Samples were rotated at room temperature (RT) for 1 hour, and washedwith PBS-Tween/1% BSA twice. 250 μl of PBS-Tween/1% BSA with 1:50dilution of anti-mouse alexa Fluro 488 was added into cells and sampleswere rotated at RT for 1 hour. Samples were washed with PBS-Tween/1% BSAtwice and then resuspended in 500 μl PBS. Flow cytometry was performedin Children's Hospital Boston.

Statistical Analysis.

Antibody and IL-17A concentrations and NP colonization densities werecompared by the Mann-Whitney U test using PRISM (version 4.0a, GraphPadSoftware, Inc). Differences in survival were analyzed with theKaplan-Meier test, using PRISM as well.

Example 1 Selection of Protein Candidates by Bioinformatic Analysis

At present, there are at least 42 S. pneumoniae sequences available fromthe integrated microbial genomes website (world wide web:“llimg.jgi.doe.gov/cgi-bin/w/main.cgi”). Beginning with the sequencedTIGR4 strain, the inventors analyzed the genome for predicted secretionsignal peptides and cell wall anchor motifs. The inventors identified335 proteins with a secretion signal peptide in TIGR4 and 15 proteinswith possible cell wall anchor motifs.

The protein library was further narrowed down to 76 proteins based onthe following parameters which were chosen a priori:

(a) Conservation across all the sequenced pneumococci (>90% identity atthe amino acid level).

(b) Exclusion of any protein that had >40% homology with proteins in thehuman genome

(c) Exclusion of proteins that contain an extracellular domain smallerthan 100 amino acids.

The breakdown of the 76 proteins is as follows: 23 hypotheticalproteins, 17 proteins that play a role in substrate binding andtransportation, 17 proteins with enzymatic activities and 19 others ofunknown or hypothetical function. A full list of the proteins and theirsequence is shown in Table 2.

TABLE 2 A list of the pneumococcal proteinsSEQ ID NO: 1-83 and the primer sequencesfor isolation and amplification. Predicted Primer Proteins* functionsnames Sequences SP0010 (23-end) hypothetical Sp0010-1 CGGGATCCGAATTCGA(SEQ ID NO: 153) protein GCTCCGAAAAAGAAGT CGTCTATAC  (SEQ ID NO: 233)Sp0010-2  CTCGAGTGCGGCCGCA AGCTTTTTTAGAACCT CATAAACATC (SEQ ID NO: 235)SP0043 (42-end) competence factor Sp0043-1  CGGGATCCGAATTCGA(SEQ ID NO: 154) transport protein GCTCCGAGAAGGAGAT ComB GAGTTTGTC(SEQ ID NO: 236) Sp0043-2 CTCGAGTGCGGCCGCA AGCTTCTCTTTGTTCA AAAATTGATC(SEQ ID NO: 237) SP0079 (24-end) potassium uptake Sp0079-1CGGGATCCGAATTCGA (SEQ ID NO: 155) protein, Trk GCTCCAAGCAGGATAT familyGAATATTATC (SEQ ID NO: 238) Sp0079-2  CTCGAGTGCGGCCGCA AGCTTCGAATTCAATGCTACTAGG (SEQ ID NO: 404) SP0084 (110-end) histidine kinase Sp0084-1CGGGATCCGAATTCGA (SEQ ID NO: 156) (EC 2.7.13.3) GCTCCTTTGATTCCTT(IMGterm) GGAAGAAAG (SEQ ID NO: 405) Sp0084-2 CTCGAGTGCGGCCGCAAGCTTGGCTTTATTTT CACTACCAG (SEQ ID NO: 242) SP0092 (30-end)carbohydrate ABC Sp0092-1 CGGGATCCGAATTCGA (SEQ ID NO: 157) transporterGCTCCAACAGCAAAAA substrate-binding AGCTGCTG protein, CUT1(SEQ ID NO: 243) family (TC Sp0092-2 CTCGAGTGCGGCCGCA 3.A.1.1.-)AGCTTTTTTTTGTTTT (IMGterm) TCAAGAATTCATC (SEQ ID NO: 244)SP0098 (30-end) hypothetical Sp0098-1 CGGGATCCGAATTCGA (SEQ ID NO: 158)protein GCTCCGACGGGATTAA GAGCCTAC (SEQ ID NO: 245) Sp0098-2CTCGAGTGCGGCCGCA AGCTTACGTCTGCTTG GTGTGGAT (SEQ ID NO: 246)SP0106 (29-end) L-serine Sp0106-1 CGGGATCCGAATTCGA (SEQ ID NO: 159)ammonia-lyase GCTCCGTTCGTATTGG (EC 4.3.1.17) GAAGATTG  (IMGterm)(SEQ ID NO: 247) Sp0106-2 CTCGAGTGCGGCCGCA AGCTTTTTAAAGAAAT TGACATTGTG(SEQ ID NO: 248) SP0107 (30-end) LysM domain Sp0107-1 CGGGATCCGAATTCGA(SEQ ID NO: 160) protein GCTCCCAAGAATCATC AACTTACAC (SEQ ID NO: 249)Sp0107-2 CTCGAGTGCGGCCGCA AGCTTATACCAGCCAT TGTTAAGCC (SEQ ID NO: 250)SP0127 (26-end) hypothetical Sp0127-1 CGGGATCCGAATTCGA (SEQ ID NO: 161)protein GCTCCGAGACGACGAT TAATATTAAG (SEQ ID NO: 251) Sp0127-2CTCGAGTGCGGCCGCA AGCTTTAGGCGTTTAA TGTAAGACTC (SEQ ID NO: 252)SP0149 (25-end) lipoprotein Sp0149-1 CGGGATCCGAATTCGA (SEQ ID NO: 162)GCTCCAACTCAGAAAA GAAAGCAGAC (SEQ ID NO: 253) Sp0149-2 CTCGAGTGCGGCCGCAAGCTTCCAAACTGGTT GATCCAAAC (SEQ ID NO: 254) SP0191 (26-end) hypotheticalSp0191-1 CGGGATCCGAATTCGA (SEQ ID NO: 163) protein GCTCCCCAGCTACAAAAACAGAAAAAG (SEQ ID NO: 255) Sp0191-2 CTCGAGTGCGGCCGCA AGCTTTTGTTCTGTCGCGCCATTTGC (SEQ ID NO: 256) SP0198 (45-end) hypothetical Sp0198-1CGGGATCCGAATTCGA (SEQ ID NO: 164) protein GCTCCAATACCAATAC TGCAAATGC(SEQ ID NO: 257) Sp0198-2 CTCGAGTGCGGCCGCA AGCTTTTTAGTTAAAA CGATTTGGTC(SEQ ID NO: 258) SP0249 (26-end) PTS system, IIB Sp0249-1CGGGATCCGAATTCGA (SEQ ID NO: 165) component GCTCCCAATCTAGTGG AGTTGAGG(SEQ ID NO: 259) Sp0249-2 CTCGAGTGCGGCCGCA AGCTTCCCACTAATCA AAGATAGG(SEQ ID NO: 260) SP0321 (1-end) PTS system, IIA Sp0321-1CGGGATCCGAATTCGA (SEQ ID NO: 14) component GCTCCATGAAAATTGT ACTTGTAG(SEQ ID NO: 261) Sp0321-2 CTCGAGTGCGGCCGCA AGCTTAATACCCGATT CGAAATCTTC(SEQ ID NO: 263) SP0346 (98-end) capsular Sp0346-1 CGGGATCCGAATTCGA(SEQ ID NO: 166) polysaccharide GCTCCGGACTGACCAA biosynthesisTCGTTTAAATG protein Cps4A (SEQ ID NO: 264) Sp0346-2 CTCGAGTGCGGCCGCAAGCTTTCTACCCTCCA TCACATCC (SEQ ID NO: 265) SP0402 (29-end)signal peptidase. Sp0402-1 CGGGATCCGAATTCGA (SEQ ID NO: 167)Serine peptidase. GCTCCTGGAGCAATGT MEROPS family TCGCGTAG S26A (IMGterm)(SEQ ID NO: 266) Sp0402-2 CTCGAGTGCGGCCGCA AGCTTAAATGTTCCGA TACGGGTG(SEQ ID NO: 267) SP0453 (25-298)  amino acid ABC Sp0453-1CGGGATCCGAATTCGA (SEQ ID NO: 168) transporter GCTCCGATGAATATTTsubstrate-binding ACGCATCG protein, PAAT (SEQ ID NO: 268) family (TCSp0453-2 CTCGAGTGCGGCCGCA 3.A.1.3.-)/amino AGCTTACCAGCACCACacid ABC transporter GCAAGAG membrane protein, (SEQ ID NO: 269)PAAT family (TC 3.A.1.3.-)(IMGterm) SP0564 (21-end) hypotheticalSp0564-1 CGGGATCCGAATTCGA (SEQ ID NO: 169) protein GCTCCTTTTCAAGTACGGTGACTAAG (SEQ ID NO: 270) Sp0564-2 CTCGAGTGCGGCCGCA AGCTTCTTGAACTTGATGCCATTTTC (SEQ ID NO: 271) SP0582 (92-end) hypothetical Sp0582-1CGGGATCCGAATTCGA (SEQ ID NO: 170) protein GCTCCGATAAAACCCT TTCTTCTGC(SEQ ID NO: 272) Sp0582-2 CTCGAGTGCGGCCGCA AGCTTAAATGTGATTT CTGTAAAAATAC(SEQ ID NO: 273) SP0589 (36-end) serine O- Sp0589-1 CGGGATCCGAATTCGA(SEQ ID NO: 171) acetyltransferase GCTCCGCCCACCGTCT (EC 2.3.1.30)CTCGCAT (IMGterm) (SEQ ID NO: 274) Sp0589-2 CTCGAGTGCGGCCGCAAGCTTCAAACCAGACG ATCTGTGAC (SEQ ID NO: 275) SP0601 (36-297)transmembrane Sp0601-1 CGGGATCCGAATTCGA (SEQ ID NO: 172) protein Vexp3GCTCCATCAAGGGAGC TACTGCCAAG (SEQ ID NO: 276) Sp0601-2 CTCGAGTGCGGCCGCAAGCTTCATACCAGAGA TAGATTGCTC (SEQ ID NO: 277) SP0604 (223-end) sensor histidine Sp0604-1 CGGGATCCGAATTCGA (SEQ ID NO: 173) kinase VncSGCTCCGAAGCCATTCT CCAGCTGG (SEQ ID NO: 278) Sp0604-2 CTCGAGTGCGGCCGCAAGCTTGTCTTGGACGA CTTTTGG (SEQ ID NO: 279) SP0617 (44-end) hypotheticalSp0617-1 CGGGATCCGAATTCGA (SEQ ID NO: 174) protein GCTCCAACGGAGATTTTCAAGGAGC (SEQ ID NO: 280) Sp0617-2 CTCGAGTGCGGCCGCA AGCTTCTCACTAGTCTCATATATTTTTC (SEQ ID NO: 281) SP0620 (27-end) amino acid ABC Sp0620-1CGGGATCCGAATTCGA (SEQ ID NO: 175) transporter GCTCCAGCGCTCAAAAsubstrate-binding GACAATCG protein, PAAT (SEQ ID NO: 282) family (TCSp0620-2 CTCGAGTGCGGCCGCA 3.A.1.3.-) AGCTTTTGTAACTGAG (IMGterm)ATTGATCTG (SEQ ID NO: 283) SP0629 (21-end) D-Ala-D-Ala Sp0629-1CGGGATCCGAATTCGA (SEQ ID NO: 176) carboxypeptidase. GCTCCCAAGAAAAAACMetallo peptidase. AAAAAATGAAG MEROPS family (SEQ ID NO: 284)M15B (IMGterm) Sp0629-2 CTCGAGTGCGGCCGCA AGCTTATCGACGTAGT CTCCGCC(SEQ ID NO: 285) SP0648 (40-776) beta-galactosidase Sp0648-1CGGGATCCGAATTCGA (SEQ ID NO: 177) (EC:3.2.1.23) GCTCCGAATCTGTAGT(IMGterm) TTATGCGG (SEQ ID NO: 286) Sp0648-2 CTCGAGTGCGGCCGCAAGCTTTGCTAATTCTT TGTTTTCC (SEQ ID NO: 287) SP0648 (777-1676) Sp0648-3CGGGATCCGAATTCGA (SEQ ID NO: 178) GCTCCTCCAAAGTAGC TGACTCAG(SEQ ID NO: 288) Sp0648-4 CTCGAGTGCGGCCGCA AGCTTAGACTCAAGGT AGTAGTCTG(SEQ ID NO: 289) SP0648 (1677-end) Sp0648-5 CGGGATCCGAATTCGA(SEQ ID NO: 179) GCTCCGTAGATGGAAA AGTTCCG (SEQ ID NO: 290) Sp0648-6CTCGAGTGCGGCCGCA AGCTTGTCTTCTTTTT TACCTTTAG (SEQ ID NO: 291)SP0659 (28-end) thioredoxin family Sp0659-1 CGGGATCCGAATTCGA(SEQ ID NO: 180) protein GCTCCGAACACCAAAC GAAAGATG (SEQ ID NO: 292)Sp0659-2 CTCGAGTGCGGCCGCA AGCTTGGCTAATTCCT TCAAAGTTTG (SEQ ID NO: 293)SP0662 (29-276) sensor histidine Sp0662-1 CGGGATCCGAATTCGA(SEQ ID NO: 181) kinase, putative GCTCCTACTATCAATC AAGTTCTTC(SEQ ID NO: 294) Sp0662-2 CTCGAGTGCGGCCGCA AGCTTGAGCTGACTCC GAACCTGGTC(SEQ ID NO: 295) SP0662 (300-end) Sp0662-3 CGGGATCCGAATTCGA(SEQ ID NO: 182) GCTCCAAACGCTGGAT TGCTCCT (SEQ ID NO: 296) Sp0662-4CTCGAGTGCGGCCGCA AGCTTGCTAGTTTCTA TTCTATTTAT (SEQ ID NO: 297)SP0664 (103-629) zinc Sp0664-1 CGGGATCCGAATTCGA (SEQ ID NO: 183)metalloprotease GCTCCACCCTAGCGCT ZmpB GGCTAGTCG (SEQ ID NO: 298)Sp0664-2 CTCGAGTGCGGCCGCA AGCTTCTCAACTTTTT TAAGATCTA (SEQ ID NO: 299)SP0664 (630-1200) Sp0664-3 CGGGATCCGAATTCGA (SEQ ID NO: 184)GCTCCCTTAAAAATAT TAAACGTAC (SEQ ID NO: 300) Sp0664-4 CTCGAGTGCGGCCGCAAGCTTGATTGCATTAA CTCTATAGTC (SEQ ID NO: 301) SP0664 (1201-end) Sp0664-5CGGGATCCGAATTCGA (SEQ ID NO: 185) GCTCCAAAGATTTATA TTTAGAAG(SEQ ID NO: 302) Sp0664-6 CTCGAGTGCGGCCGCA AGCTTTTTAAAGATTG AAGTTTTAAAGC(SEQ ID NO: 303) SP0678 (23-end) hypothetical Sp0678-1 CGGGATCCGAATTCGA(SEQ ID NO: 186) protein GCTCCCGTATTCGCCG TGCGGCTA (SEQ ID NO: 304)Sp0678-2 CTCGAGTGCGGCCGCA AGCTTGCTAGTCTTCA CTTTCC (SEQ ID NO: 305)SP0724 (35-end) hydroxyethylthiazole Sp0724-1 CGGGATCCGAATTCGA(SEQ ID NO: 187) kinase, GCTCCGATGATTCCCG putative TGAAGTTC(SEQ ID NO: 306) Sp0724-2 CTCGAGTGCGGCCGCA AGCTTTTCATAAACCT CTCCTTTG(SEQ ID NO: 307) SP0742 (43-end) hypothetical Sp0742-1 CGGGATCCGAATTCGA(SEQ ID NO: 188) protein GCTCCGCTGATCAGGT CTTTGTTG (SEQ ID NO: 308)Sp0742-2 CTCGAGTGCGGCCGCA AGCTTATCAATTTCAT AGCCCATCAG (SEQ ID NO: 309)SP0757 (44-451) cell division Sp0757-1 CGGGATCCGAATTCGA (SEQ ID NO: 189)protein FtsX GCTCCATTTTCAATAC (IMGterm) AGCGAAAC (SEQ ID NO: 310)Sp0757-2 CTCGAGTGCGGCCGCA AGCTTAAATGAAGCTA ACTTGAAGAG (SEQ ID NO: 311)SP0785 (33-end) hypothetical Sp0785-1 CGGGATCCGAATTCGA (SEQ ID NO: 190)protein GCTCCTTTAGACAACC TTCTCAGAC (SEQ ID NO: 312) Sp0785-2CTCGAGTGCGGCCGCA AGCTTATTAGTTGCTT CATCAGCC (SEQ ID NO: 313)SP0787 (43-290) hypothetical Sp0787-1 CGGGATCCGAATTCGA (SEQ ID NO: 191)protein GCTCCTCTCGTCAAGT CAATAAAG (SEQ ID NO: 314) Sp0787-2CTCGAGTGCGGCCGCA AGCTTCGTCGTCATAA AACTAAACG (SEQ ID NO: 315)SP0872 (30-end) D,D- Sp0872-1 CGGGATCCGAATTCGA (SEQ ID NO: 192)carboxypeptidase GCTCCAAACATGCGAT PBP3. Serine TGCTGTTG peptidase.(SEQ ID NO: 316) MEROPS family Sp0872-2 CTCGAGTGCGGCCGCA S11 (IMGterm)AGCTTTAATTTCTCGT TAACAAAGCG (SEQ ID NO: 317) SP0878 (245-end)SpoE family Sp0878-1 CGGGATCCGAATTCGA (SEQ ID NO: 193) proteinGCTCCACAGAGGAAGC TGTTCAAAATC (SEQ ID NO: 318) Sp0878-2 CTCGAGTGCGGCCGCAAGCTTTTGTTGTAACA CTTTTCGAGG (SEQ ID NO: 319) SP0899 (31-end)hypothetical Sp0899-1 CGGGATCCGAATTCGA (SEQ ID NO: 194) proteinGCTCCGAGGGGACGAA TCAAAGGC (SEQ ID NO: 320) Sp0899-2 CTCGAGTGCGGCCGCAAGCTTAAGTTTAACCC ACTTATCATTATC (SEQ ID NO: 321) SP1002 (22-end) adhesionSp1002-1 CGGGATCCGAATTCGA (SEQ ID NO: 195) lipoprotein GCTCCGGTCAAAAGGAAAGTCAGAC (SEQ ID NO: 322) Sp1002-2 CTCGAGTGCGGCCGCA AGCTTCTTTAATTCTTCTGCTAGAATAC (SEQ ID NO: 323) SP1026 (24-end) hypothetical Sp1026-1CGGGATCCGAATTCGA (SEQ ID NO: 196) protein GCTCCGTTCATCAAGA TGTCAAAC(SEQ ID NO: 324) Sp1026-2 CTCGAGTGCGGCCGCA AGCTTGCCAGATGTTG AAAAGAGAG(SEQ ID NO: 325) SP1032 (22-end) iron-compound Sp1032-1 CGGGATCCGAATTCGA(SEQ ID NO: 197) ABC transporter, GCTCCTCTAATTCTGT iron compound-TAAAAATGAAG binding protein (SEQ ID NO: 326) Sp1032-2 CTCGAGTGCGGCCGCAAGCTTTTTCGCATTTT TGCATGCATTTC (SEQ ID NO: 327) SP1069 (34-end)hypothetical Sp1069-1 CGGGATCCGAATTCGA (SEQ ID NO: 198) proteinGCTCCTCTTCAATGAA TAAATCAG (SEQ ID NO: 328) Sp1069-2 CTCGAGTGCGGCCGCAAGCTTTTCGATGACTT GTCCTGCTTC (SEQ ID NO: 329) SP1154 (155-694)IgA1-specific Sp1154-1 CGGGATCCGAATTCGA (SEQ ID NO: 199)metallopeptidase. GCTCCGAAAATCATCT Metallo peptidase. TTTGCTAAATTACMEROPS family (SEQ ID NO: 330) M26 (IMGterm) Sp1154-2 CTCGAGTGCGGCCGCAAGCTTTGTGTTTGATT CGGTTGAAAC (SEQ ID NO: 331) SP1154 (695-1374) Sp1154-3CGGGATCCGAATTCGA (SEQ ID NO: 200) GCTCCTCCAATTCAAA TGGAAACG(SEQ ID NO: 332) Sp1154-4 CTCGAGTGCGGCCGCA AGCTTACCAAAGAAGT CCAAATGG(SEQ ID NO: 333) SP1154 (1375-end) Sp1154-5 CGGGATCCGAATTCGA(SEQ ID NO: 201) GCTCCAAGGGGAATGC TTCACCATTAG (SEQ ID NO: 334) Sp1154-6CTCGAGTGCGGCCGCA AGCTTTTTTTTATTCT CAAAAATTG (SEQ ID NO: 335)SP1267 (25-end) licC protein Sp1267-1 CGGGATCCGAATTCGA (SEQ ID NO: 202)GCTCCTTGGTTCAGGT TAATCAAAAAC (SEQ ID NO: 336) Sp1267-2 CTCGAGTGCGGCCGCAAGCTTATTTTCGTTTT TAAGAATTTC (SEQ ID NO: 337) SP1376 (32-end) shikimateSp1376-1 CGGGATCCGAATTCGA (SEQ ID NO: 203) dehydrogenaseGCTCCGCGACAGCTAC (EC 1.1.1.25) CAACGGTG (IMGterm) (SEQ ID NO: 338)Sp1376-2 CTCGAGTGCGGCCGCA AGCTTTTGGTATTTTT CTGTTAAAG (SEQ ID NO: 339)SP1386 (33-end) spermidine/ Sp1386-1 CGGGATCCGAATTCGA (SEQ ID NO: 204)putrescine ABC GCTCCGATAGTCAAAA transporter, ATTGGTTATC spermidine/(SEQ ID NO: 340) putrescine-binding Sp1386-2 CTCGAGTGCGGCCGCA proteinAGCTTCTTCCGATACA TTTTAAACTG (SEQ ID NO: 341) SP1404 (31-end)hypothetical Sp1404-1 CGGGATCCGAATTCGA (SEQ ID NO: 205) proteinGCTCCGCCTATGAAGG CAAAGTAG (SEQ ID NO: 342) Sp1404-2 CTCGAGTGCGGCCGCAAGCTTCTTTCCAAGAG AAATCTTTC (SEQ ID NO: 343) SP1405 (19-end)transcriptional Sp1405-1 CGGGATCCGAATTCGA (SEQ ID NO: 206) regulator SpxGCTCCTGGTTAGAAAA ACATAAGG (SEQ ID NO: 344) Sp1405-2 CTCGAGTGCGGCCGCAAGCTTCTAATACCAGC TCTCATTC (SEQ ID NO: 345) SP1419 (27-end)acetyltransferase, Sp1419-1 CGGGATCCGAATTCGA (SEQ ID NO: 207)GNAT family GCTCCATGTTTCAAAA TTGGGCTTC (SEQ ID NO: 346) Sp1419-2CTCGAGTGCGGCCGCA AGCTTACATTCTTCCC TACTTATACC (SEQ ID NO: 347)SP1479 (40-end) peptidoglycan N- Sp1479-1 CGGGATCCGAATTCGA(SEQ ID NO: 208) acetylglucosamine GCTCCAAGATCTACCA deacetylase AGCAAAAAAG (SEQ ID NO: 348) Sp1479-2 CTCGAGTGCGGCCGCA AGCTTTTCATCACGACTATAGTACAGC (SEQ ID NO: 349) SP1500 (27-end) amino acid ABC Sp1500-1CGGGATCCGAATTCGA (SEQ ID NO: 209) transporter GCTCCACTAGTGGAGAsubstrate-binding TAATTGGTC protein, PAAT (SEQ ID NO: 350) family (TCSp1500-2 CTCGAGTGCGGCCGCA 3.A.1.3.-) AGCTTCTGTCCTTCTT (IMGterm)TTACTTCTTTG (SEQ ID NO: 351) SP1545 (29-end) hypothetical Sp1545-1CGGGATCCGAATTCGA (SEQ ID NO: 210) protein GCTCCGAAGGAGAAAA ATTAGCTC(SEQ ID NO: 352) Sp1545-2 CTCGAGTGCGGCCGCA AGCTTTAGGCCCTCCT TGTTGACC(SEQ ID NO: 353) SP1560 (28-end) hypothetical Sp1560-1 CGGGATCCGAATTCGA(SEQ ID NO: 211) protein GCTCCCAAAACAGTAC CAGTGCTAG (SEQ ID NO: 354)Sp1560-2 CTCGAGTGCGGCCGCA AGCTTATTCGTTTTTG AACTAGTTGC (SEQ ID NO: 355)SP1624 (1-217) 1-acyl-sn- Sp1624-1 CGGGATCCGAATTCGA (SEQ ID NO: 212)glycerol-3- GCTCCATGTTTTATAC phosphate TTATTTGCGTG acyltransferase(SEQ ID NO: 356) (EC 2.3.1.51) Sp1624-2 CTCGAGTGCGGCCGCA (IMGterm)AGCTTGGCAGGGATGC GGATAAACC (SEQ ID NO: 357) SP1652 (62-397) hypotheticalSp1652-1 CGGGATCCGAATTCGA (SEQ ID NO: 213) protein GCTCCAAAGTAACCAGTCCCAACATGG (SEQ ID NO: 358) Sp1652-2 CTCGAGTGCGGCCGCA AGCTTACTGGATGAAGCATTGCTATAC (SEQ ID NO: 359) SP1683 (65-end) carbohydrate ABC Sp1683-1CGGGATCCGAATTCGA (SEQ ID NO: 214) transporter GCTCCAAATCAATCATsubstrate-binding CGAAGCGTTTG protein, CUT1 (SEQ ID NO: 360) family (TCSp1683-2 CTCGAGTGCGGCCGCA 3.A.1.1.-) AGCTTTTGTTTCATAG (IMGterm)CTTTTTTGATTG (SEQ ID NO: 361) SP1826 (36-end) ABC transporter, Sp1826-1CGGGATCCGAATTCGA (SEQ ID NO: 215) substrate-binding GCTCCATGCCTAATTAprotein TAAATTTGTTG  (SEQ ID NO: 362) Sp1826-2 CTCGAGTGCGGCCGCAAGCTTTTTTCTACCCT CCTTTTCC (SEQ ID NO: 363) SP1872 (40-end) iron-compoundSp1872-1 CGGGATCCGAATTCGA (SEQ ID NO: 216) ABC transporter,GCTCCGAAGAAAAAGC iron-compound- TGATAAAAGTC binding protein(SEQ ID NO: 364) Sp1872-2 CTCGAGTGCGGCCGCA AGCTTGCTGAATTAGA ATACGTACAA(SEQ ID NO: 365) SP1891 (40-end) oligopeptide ABC Sp1891-1CGGGATCCGAATTCGA (SEQ ID NO: 217) transporter, GCTCCACAGAGGTAAColigopeptide- CATTAAAAG binding protein (SEQ ID NO: 366) AmiA Sp1891-2CTCGAGTGCGGCCGCA AGCTTTTTCAAAGCTT TTTGTATGTC (SEQ ID NO: 367)SP1897 (30-end) multiple sugar- Sp1897-1 CGGGATCCGAATTCGA(SEQ ID NO: 218) binding protein GCTCCGCGGATGGCAC (IMGterm) AGTGACC(SEQ ID NO: 368) Sp1897-2 CTCGAGTGCGGCCGCA AGCTTATCCACATCCG CTTTCATC(SEQ ID NO: 369) SP1942 (37-end) transcriptional Sp1942-1CGGGATCCGAATTCGA (SEQ ID NO: 219) regulator, putative GCTCCAAAACCTATAAAAAAATCGGTG (SEQ ID NO: 370) Sp1942-2 CTCGAGTGCGGCCGCA AGCTTATTATCTTCATCACCAACAGG (SEQ ID NO: 371) SP1966 (25-end) UDP-N- Sp1966-1CGGGATCCGAATTCGA (SEQ ID NO: 220) acetylglucosamine GCTCCGCAGTCTTACC 1-CTTGTTGGC carboxyvinyltransferase (SEQ ID NO: 372) (EC 2.5.1.7) Sp1966-2CTCGAGTGCGGCCGCA (IMGterm) AGCTTTTCATCTTCAT CACTTGCC (SEQ ID NO: 373)SP1967 (30-end); hypothetical Sp1967-1 CGGGATCCGAATTCGA (SEQ ID NO: 221)protein GCTCCATAGAGGTTCC AGGTGGTTCG (SEQ ID NO: 374) Sp1967-2CTCGAGTGCGGCCGCA AGCTTGGGATTGTTTT TCAAGTAATC (SEQ ID NO: 375)SP1998 (51-end) asparaginase (EC Sp1998-1 CGGGATCCGAATTCGA(SEQ ID NO: 222) 3.5.1.1) GCTCCGGGATTGTTTT (IMGterm) TCAAGTAATC(SEQ ID NO: 376) Sp1998-2 CTCGAGTGCGGCCGCA AGCTTGCCTTCCATAT AGTCTTTC(SEQ ID NO: 377) SP2048 (40-end) hypothetical Sp2048-1 CGGGATCCGAATTCGA(SEQ ID NO: 223) protein GCTCCAGTCAGCTCCT CATTTCAG (SEQ ID NO: 378)Sp2048-2 CTCGAGTGCGGCCGCA AGCTTACTTTTTTCTT TTTCCACAC (SEQ ID NO: 379)SP2050 (35-end) competence Sp2050-1 CGGGATCCGAATTCGA (SEQ ID NO: 224)protein Cg1D GCTCCGTAGAGGAACA GATTTTCT (SEQ ID NO: 380) Sp2050-2CTCGAGTGCGGCCGCA AGCTTATTTTTTGTTT CCTTAATGCG (SEQ ID NO: 381)SP2083 (192-end) sensor histidine Sp2083-1 CGGGATCCGAATTCGA(SEQ ID NO: 225) kinase PnpS GCTCCTTTAGCCCCAC CCAATCTGTG(SEQ ID NO: 382) Sp2083-2 CTCGAGTGCGGCCGCA AGCTTGTCCTGTGCGA AAGATTGG(SEQ ID NO: 383) SP2084 (30-end) phosphate ABC Sp2084-1 CGGGATCCGAATTCGA(SEQ ID NO: 226) transporter GCTCCAAACAGTCAGC substrate-bindingTTCAGGAAC protein, PhoT (SEQ ID NO: 384) family (TC Sp2084-2CTCGAGTGCGGCCGCA 3.A.1.7.1) AGCTTTTTAATCTTGT (IMGterm) CCCAGGTGG(SEQ ID NO: 385) SP2088 (30-end); phosphate uptake Sp2088-1CGGGATCCGAATTCGA (SEQ ID NO: 227) regulator, PhoU GCTCCGCCTTACTGGC(IMGterm) CTTAGCCTCC (SEQ ID NO: 386) Sp2088-2 CTCGAGTGCGGCCGCAAGCTTATTCAAATCCA CTAGTTCTC (SEQ ID NO: 387) SP2145 (1-end)antigen, cell wall Sp2145-1 CGGGATCCGAATTCGA (SEQ ID NO: 70)surface anchor GCTCCATGAAACCACT family ACTTGAAACC (SEQ ID NO: 388)Sp2145-2 CTCGAGTGCGGCCGCA AGCTTGTGACTTGGTA ACCAGCTG (SEQ ID NO: 389)SP2151 (25-end) carbamate kinase Sp2151-1 CGGGATCCGAATTCGA(SEQ ID NO: 228) (EC 2.7.2.2) GCTCCCAACAAGAAGC (IMGterm) TTTAGTTG(SEQ ID NO: 390) Sp2151-2 CTCGAGTGCGGCCGCA AGCTTTCCTTTTTCAA TAATTGTTCC(SEQ ID NO: 391) SP2187 (32-end); hypothetical Sp2187-1 CGGGATCCGAATTCGA(SEQ ID NO: 229) protein GCTCCGTAATGGAAGA AACAGGAT (SEQ ID NO: 392)Sp2187-2 CTCGAGTGCGGCCGCA AGCTTAGTGAATAATA ACTGGCGAATC (SEQ ID NO: 393)SP2192 (224-end) sensor histidine Sp2192-1 CGGGATCCGAATTCGA(SEQ ID NO: 230) kinase GCTCCGAGCAAATTGT AAAATTGC (SEQ ID NO: 394)Sp2192-2 CTCGAGTGCGGCCGCA AGCTTCAAGCTAATCT TAAATTCC (SEQ ID NO: 395)SP2197 (30-end) ABC transporter, Sp2197-1 CGGGATCCGAATTCGA(SEQ ID NO: 231) substrate-binding GCTCCAAAGACAACAA protein, putativeAGAGGCAGAAC (SEQ ID NO: 396) Sp2197-2 CTCGAGTGCGGCCGCA AGCTTTTTCACAAATTCGTTGGTGAAG (SEQ ID NO: 397) SP2207 (30-end) competence Sp2207-1CGGGATCCGAATTCGA (SEQ ID NO: 232) protein ComF, GCTCCTGTTCAGACTGputative TGATTCTAC (SEQ ID NO: 398) Sp2207-2 CTCGAGTGCGGCCGCAAGCTTTCTTACAAGGG AAAATGTT (SEQ ID NO: 399) SP2218 (106-end) rod shape-Sp2218-1 CGGGATCCGAATTCGA (SEQ ID NO: 234) determining GCTCCTCTAAATTGCAprotein MreC AGCCACAAAG (IMGterm) (SEQ ID NO: 400) Sp2218-2CTCGAGTGCGGCCGCA AGCTTTGAATTCCCCA CTAATTCTATC (SEQ ID NO: 401) *Aminoacid residues used for cloning; end refers to C-terminus.

Example 2 Screening Antigens from Mouse Splenocytes and Human PeripheralBlood Mononuclear Cells (PBMC)

60 proteins out of 80 genetic constructs were successfully cloned andexpressed in, then purified from, E. coli. These proteins were then usedin stimulation experiments with either mouse splenocytes or human PBMCto evaluate the ability of these proteins to recall a potent IL-17Aresponse (elicitation of IL-17A responses is a predictor ofimmunogenicity and protection against colonization by a pneumococcalantigen—(see Moffitt K L, Gierahn T M, Lu Y J, et al. T(H)17-BasedVaccine Design for Prevention of Streptococcus pneumoniae Colonization.Cell Host Microbe 2011; 9:158-65).

The mouse splenocytes were obtained from three sources: 1. Micepreviously immunized with a whole cell vaccine (WCV); 2. Mice previouslycolonized with a single strain of S. pneumoniae for 10 days and 3. Micewere sequentially colonized with S. pneumoniae serotype 6B, 14F and 19Fstrains. These three different models were used to identify putativeprotective surface antigens, as the inventors assessed if these antigensare sufficiently expressed during colonization to elicit a response byimmune cells following colonization. Furthermore, the inventors assessedif some of the surface proteins would also confer protection againstcolonization and/or invasive disease in mice. Any given protein mayprovide protection against colonization, invasive disease, or both;since both colonization and invasive disease prevention are importantgoals of vaccination, the evaluation of the ability to confer protectionin either model was included. The inventors also assessed if humans—whoare all naturally exposed to pneumococci during their lifetime—were alsocapable of responding to the proteins, as more evidence that thisprotein may be expressed during human colonization. The responses toeach protein in the different screens are summarized in Table 3 (whichalso includes whether the protein is protective against colonization, tobe discussed below).

TABLE 3 Summary of IL-17A response to protein stimulation and protectionagainst colonization. Mouse IL-17A Human IL-17A (responses to (responsesto Protection Proteins 0.2/1/5 μg of protein)* 1/10 μg of protein)* inmice SP0346 +/++/++ −/− No SP0648 +/++/+ −/− No SP0785 +/−/++ +/+ YesSP0787 +++/−/− +/− No SP1154 ++/+++/− −/− No SP1376 −/−/++ −/+ No SP1500+/−/− +++/− Yes SP1545 −/−/− ++/− No SP1872 −/−/− +++/− No SP1897 +/−/−++/+ No SP1942 +/−/++ +/− No SP2145 −/−/+ +/− No SP2151 ++/++/+++ −/− NoSP2207 +/++/+ +/− No SP0043 +/−/− −/− ND** SP0079 +/+++/− −/− ND**SP0084 +/++/− −/+ ND** SP0092 +/+/− −/+ ND** SP0098 +/+++/++ −/− ND**SP0149 −/−/− +/+ ND** SP0191 +/−/− −/+ ND** SP0198 −/+/− +/− ND** SP0249−/++/+++ −/+ ND** SP0321 −/+/− −/− ND** SP0402 −/−/− −/− ND** SP0453++/+++/− ND** ND** SP0564 +/−/− ++/− ND** SP0582 +/+++/− −/− ND** SP0601−/−/− +/− ND** SP0604 −/−/+ +/− ND** SP0617 −/−/− +/+ ND** SP0620 −/++/−+/− ND** SP0629 −/−/− +/+ ND** SP0659 −/−/− +/− ND** SP0662 +/−/− +/−ND** SP0678 −/+/− −/− ND** SP0757 +/−/++ −/− ND** SP0878 −/−/− −/− ND**SP0899 ++/−/− +/− ND** SP1002 +/+/− −/− ND** SP1032 +/−/+ ++/− ND**SP1069 +/+/− +/− ND** SP1386 −/−/+ +/− ND** SP1404 +/−/− −/− ND** SP1479−/−/− −/− ND** SP1560 −/−/− +++/− ND** SP1652 −/−/− ++/− ND** SP1683+/+/+ −/− ND** Sp1826 −/−/− ND** ND** SP2084 +/+/+ +/− ND** SP2192 −/−/−+/− ND** SP2197 +/−/− +++/− ND** SP2218 +/−/− −/+ ND** *IL-17Aproduction in response to protein stimulation: −, <25 pg/ml, +, >25pg/ml, ++, >100 pg/ml, +++, >250 pg/ml, **ND, not determined

Example 3 SP0785 and SP1500 Provide Protection Against Colonization

The inventors then tested the 14 antigens to see if they could provideprotection in a mouse colonization model. For each protein tested, 5 mgof each antigen were mixed with adjuvant CT and used to immunize micetwice weekly. Mice were challenged with a serotype 6B pneumococcalstrain and the protection was accessed 7 days later for pneumococcalcolonization in the nose. Two proteins, SP0785 and SP1500 showedsignificant protection compared to the adjuvant alone group (p=0.0023and p=0.0009 for SP0785 and SP1500, respectively) (FIG. 1 ). The other12 antigens tested were not protective in this colonization model.

Example 4 Protection Against Sepsis Challenge

At the same time, the inventors evaluated whether these proteins couldconfer protection, when used as immunogens, against invasive disease.Groups (n=10 mice per group) of C57/BL6 mice were immunized with one ofthe 15 proteins subcutaneously with aluminum hydroxide as adjuvant andthen challenged in an intraperitoneal (IP) infection model with anengineered WU2 strain (wild type ply replaced with a tagged ply). Thisinfection leads to 80% death of control mice starting at 3-4 days postinfection. As summarized in Table 4, protein SP0346 protected 60% of theimmunized mice compared to 20% of the alum group (p=0.0254). Fourproteins protected 50% of mice, such as SP1386, SP1500, SP0084 andSP1479. The protection from the remaining 10 proteins was lower than50%.

TABLE 4 Protection against sepsis following immunization by thesubcutaneous route, using aluminum hydroxide as adjuvant. ProteinsSurvival rate (survival/total) None 2/10 SP0785 3/10 SP2145 1/10 SP18263/10 SP0191 3/10 SP0198 4/10 SP0564 4/10 Sp1069 2/10 SP1942 3/10 SP21513/10 SP2197 0/10 SP1386 5/10 SP1500 5/10 SP0084 5/10 SP1479 5/10 SP03466/10

Example 5 Protection Against Sepsis by Proteins Fusing to PdT

The inventors have previously shown that immunization with a fusionconjugate, in which a pneumococcal protein is genetically fused to thepneumococcal pneumolysoid PdT and then covalently coupled to apolysaccharide, can enhance immune responses and protection (Lu Y J etal., Protection against Pneumococcal colonization and fatal pneumonia bya trivalent conjugate of a fusion protein with the cell wallpolysaccharide. Infect Immun 2009; 77:2076-83.; Lu et al., A bivalentvaccine to protect against Streptococcus pneumoniae and Salmonellatyphi. Vaccine 2012; 30:3405-12). The inventors next tested someantigens in fusion proteins with PdT and also fusion protein conjugatedto the Salmonella typhi polysaccharide, Vi (our nomenclature for afusion protein of protein X and PdT, then conjugated to Vi is X-PdT-Vi).As shown in FIG. 2 , immunization with a fusion protein consisting ofSP0785-PdT protected 80% mice against sepsis and fusion proteinSP2145-PdT protected 90%. The protection is not due to PdT alone, whichonly protects 30% of the infected mice, not statistically different fromthe mortality rate of control mice. Fusion proteins SP0785-PdT,SP2145-PdT and SP1500-PdT were conjugated to Vi polysaccharide asdescribed before. These mice made high level of IL-17A when stimulatedwith whole cell vaccine (WCV) (FIG. 3A), which raises the strongpossibility that these two constructs may protect against bothpneumococcal colonization and disease. FIG. 3B shows that these fusionconjugates also elicit robust antibodies to the Vi polysaccharide,predicting protection against S. typhi as well.

Similarly to fusion proteins, SP2145-PdT-Vi and SP0785-PdT-Visignificantly protected mice when compared to immunization with alumalone (80% survival in either group compared to 20% survival in alumgroup, P<0.006 for either comparison; FIG. 4 ).

Example 6 Sera from Mice Immunized with SP0785 Binds to an EncapsulatedTigr4 Strain

A flow cytometric assay was performed to test whether antibodies againstthese antigens (SP0785, SP1500, SP2145) can bind to the surface ofencapsulated pneumococcal strain. The type 4 clinical isolate TIGR4strain was cultured to late-log phase and heat fixed. Binding of mouseanti-sera was detected by FITC labeled anti-mouse IgG. As shown in FIG.5 , serum from mice immunized with SP0785 (black) can label the bacteriawhen compared to serum obtained from mice immunized with alum alone(grey), which indicates that anti-SP0785 antibody is able to bind toencapsulated pneumococcal strain.

In summary, the inventors have identified two proteins SP0785 and SP1500that protect against pneumococcal colonization, and SP0346 that protectsagainst sepsis. Fusion proteins SP0785-PdT and SP2145-PdT or theconjugation of these two proteins to Vi protected mice against sepsischallenge.

Example 7

MAPS complexes were made using biotinylated type-1 pneumococcalpolysaccharide attached to a fusion protein consisting of rhizavidin andSP0785, SP1500, SP0435 or PdT. C57/BL6 mice were immunized with amixture of 3 MAPS complexes containing SP0785, SP1500 and PdT, or amixture of all 4 MAPS complexes described above, on aluminum hydroxide,at a dosage of 6.7 μg of each antigen. Control mice received eitheraluminum hydroxide alone (alum, negative control) or a whole cellvaccine in alum (as a positive control). Immunization was givensubcutaneously three times, two weeks apart. Blood was drawn after thethird immunization and stimulated with 10 μg/ml of S. pneumoniae wholecell antigen or 5 μg/ml of purified SP0785, SP1500 or PdT protein.Antigen-specific or whole cell (WCB) IL-17A production was measured 7days post stimulation by ELISA (FIG. 6A). One week after bleeding, micewere challenged with pneumococcal 603B strain and bacterial colonizationrate in the nose was determined 10 days post challenge (FIG. 6B). Micethat received immunizations with either 3 or 4 MAPS complexes wereprotected against pneumococcal colonization.

C57BL/6 mice were immunized with vaccines containing 5 μg of proteinantigen (a fusion protein of SP0785, SP1500 and PdT conjugated to Vipolysaccharide of Salmonella typhi) adsorbed onto alum, subcutaneouslythree times, two weeks apart. Control mice received alum alone. Twoweeks after last immunization, mice were intranasally challenged withpneumococcal 603B strain and bacterial colonization rate in the nose wasdetermined by nasal wash. The mice that were immunized with the fusionconjugate were significantly protected against pneumococcal colonizationcompared to the mice in the control group (FIG. 7 ).

New Zealand white rabbits were immunized with purified SP0785, SP1500 orPdT. An equal mixture of each serum from rabbits pre-immunization vs.post immunization (in this case, 67 μl each) was given to groups of 10mice via the intraperitoneal route. Mice were then intraperitoneallyinfected with a serotype 3 strain 24 hours later and mice survival wasmonitored for 8 days. Group received post immunization serum had 60%survival comparing to 0% survival in the pre-serum group, a differencethat was highly statistically significant, attesting to the protectivecapacity of antibodies to these proteins for pneumococcal invasivedisease (FIG. 8 ).

REFERENCES

All references described herein are incorporated herein in theirentirety by reference.

-   1. Moffitt K L, Gierahn T M, Lu Y J, et al. T(H)17-Based Vaccine    Design for Prevention of Streptococcus pneumoniae Colonization. Cell    Host Microbe 2011; 9:158-65.-   2. Lu Y J, Forte S, Thompson C M, Anderson P W, Malley R. Protection    against Pneumococcal colonization and fatal pneumonia by a trivalent    conjugate of a fusion protein with the cell wall polysaccharide.    Infect Immun 2009; 77:2076-83.-   3. Lu Y J, Zhang F, Sayeed S, et al. A bivalent vaccine to protect    against Streptococcus pneumoniae and Salmonella typhi. Vaccine 2012;    30:3405-12.

Sequences:

Pneumococcal Protein SEQ ID DNA SEQ ID antigen name NO: NO: SP0010 SEQID NO: 1 SEQ ID NO: 77 SP0043 SEQ ID NO: 2 SEQ ID NO: 78 SP0079 SEQ IDNO: 3 SEQ ID NO: 79 SP0084 SEQ ID NO: 4 SEQ ID NO: 80 SP0092 SEQ ID NO:5 SEQ ID NO: 81 SP0098 SEQ ID NO: 6 SEQ ID NO: 82 SP0106 SEQ ID NO: 7SEQ ID NO: 83 SP0107 SEQ ID NO: 8 SEQ ID NO: 84 SP0127 SEQ ID NO: 9 SEQID NO: 85 SP0149 SEQ ID NO: 10 SEQ ID NO: 86 SP0191 SEQ ID NO: 11 SEQ IDNO: 87 SP0198 SEQ ID NO: 12 SEQ ID NO: 88 SP0249 SEQ ID NO: 13 SEQ IDNO: 89 SP0321 SEQ ID NO: 14 SEQ ID NO: 90 SP0346 SEQ ID NO: 15 SEQ IDNO: 91 SP0402 SEQ ID NO: 16 SEQ ID NO: 92 SP0453 SEQ ID NO: 17 SEQ IDNO: 93 SP0564 SEQ ID NO: 18 SEQ ID NO: 94 SP0582 SEQ ID NO: 19 SEQ IDNO: 95 SP0589 SEQ ID NO: 20 SEQ ID NO: 96 SP0601 SEQ ID NO: 21 SEQ IDNO: 97 SP0604 SEQ ID NO: 22 SEQ ID NO: 98 SP0617 SEQ ID NO: 23 SEQ IDNO: 99 SP0620 SEQ ID NO: 24 SEQ ID NO: 100 SP0629 SEQ ID NO: 25 SEQ IDNO: 101 SP0648 SEQ ID NO: 26 SEQ ID NO: 102 SP0659 SEQ ID NO: 27 SEQ IDNO: 103 SP0662 SEQ ID NO: 28 SEQ ID NO: 104 SP0664 SEQ ID NO: 29 SEQ IDNO: 105 SP0678 SEQ ID NO: 30 SEQ ID NO: 106 SP0724 SEQ ID NO: 31 SEQ IDNO: 107 SP0742 SEQ ID NO: 32 SEQ ID NO: 108 SP0757 SEQ ID NO: 33 SEQ IDNO: 109 SP0785 SEQ ID NO: 34 SEQ ID NO: 110 SP0787 SEQ ID NO: 35 SEQ IDNO: 111 SP0872 SEQ ID NO: 36 SEQ ID NO: 112 SP0878 SEQ ID NO: 37 SEQ IDNO: 113 SP0899 SEQ ID NO: 38 SEQ ID NO: 114 SP1002 SEQ ID NO: 39 SEQ IDNO: 115 SP1026 SEQ ID NO: 40 SEQ ID NO: 116 SP1032 SEQ ID NO: 41 SEQ IDNO: 117 SP1069 SEQ ID NO: 42 SEQ ID NO: 118 SP1154 SEQ ID NO: 43 SEQ IDNO: 119 SP1267 SEQ ID NO: 44 SEQ ID NO: 120 SP1376 SEQ ID NO: 45 SEQ IDNO: 121 SP1386 SEQ ID NO: 46 SEQ ID NO: 122 SP1404 SEQ ID NO: 47 SEQ IDNO: 123 SP1405 SEQ ID NO: 48 SEQ ID NO: 124 SP1419 SEQ ID NO: 49 SEQ IDNO: 125 SP1479 SEQ ID NO: 50 SEQ ID NO: 126 SP1500 SEQ ID NO: 51 SEQ IDNO: 127 SP1545 SEQ ID NO: 52 SEQ ID NO: 128 SP1560 SEQ ID NO: 53 SEQ IDNO: 129 SP1624 SEQ ID NO: 54 SEQ ID NO: 130 SP1652 SEQ ID NO: 55 SEQ IDNO: 131 SP1683 SEQ ID NO: 56 SEQ ID NO: 132 SP1826 SEQ ID NO: 57 SEQ IDNO: 133 SP1872 SEQ ID NO: 58 SEQ ID NO: 134 SP1891 SEQ ID NO: 59 SEQ IDNO: 135 SP1897 SEQ ID NO: 60 SEQ ID NO: 136 SP1942 SEQ ID NO: 61 SEQ IDNO: 137 SP1966 SEQ ID NO: 62 SEQ ID NO: 138 SP1967 SEQ ID NO: 63 SEQ IDNO: 139 SP1998 SEQ ID NO: 64 SEQ ID NO: 140 SP2048 SEQ ID NO: 65 SEQ IDNO: 141 SP2050 SEQ ID NO: 66 SEQ ID NO: 142 SP2083 SEQ ID NO: 67 SEQ IDNO: 143 SP2084 SEQ ID NO: 68 SEQ ID NO: 144 SP2088 SEQ ID NO: 69 SEQ IDNO: 145 SP2145 SEQ ID NO: 70 SEQ ID NO: 146 SP2151 SEQ ID NO: 71 SEQ IDNO: 147 SP2187 SEQ ID NO: 72 SEQ ID NO: 148 SP2192 SEQ ID NO: 73 SEQ IDNO: 149 SP2197 SEQ ID NO: 74 SEQ ID NO: 150 SP2207 SEQ ID NO: 75 SEQ IDNO: 151 SP2218 SEQ ID NO: 76 SEQ ID NO: 152SEQ ID NO: 153/SP0010 (23-end)SEQ ID NO: 154/SP0043 (42-end)SEQ ID NO: 155/SP0079 (24-end)SEQ ID NO: 156/SP0084 (110-end)SEQ ID NO: 157/SP0092 (30-end)SEQ ID NO: 158/SP0098 (30-end)SEQ ID NO: 159/SP0106 (29-end)SEQ ID NO: 160/SP0107 (30-end)SEQ ID NO: 161/SP0127 (26-end)SEQ ID NO: 162/SP0149 (25-end)SEQ ID NO: 163/SP0191 (26-end)SEQ ID NO: 164/SP0198 (45-end)SEQ ID NO: 165/SP0249 (26-end)SEQ ID NO: 14/SP0321 (1-end)SEQ ID NO: 166/SP0346 (98-end)SEQ ID NO: 167/SP0402 (29-end)SEQ ID NO: 168/SP0453 (25-298)SEQ ID NO: 169/SP0564 (21-end)SEQ ID NO: 170/SP0582 (92-end)SEQ ID NO: 171/SP0589 (36-end)SEQ ID NO: 172/SP0601 (36-297)SEQ ID NO: 173/SP0604 (223-end)SEQ ID NO: 174/SP0617 (44-end)SEQ ID NO: 175/SP0620 (27-end)SEQ ID NO: 176/SP0629 (21-end)SEQ ID NO: 177/SP0648 (40-776)SEQ ID NO: 178/SP0648 (777-1676)SEQ ID NO: 179/SP0648 (1677-end)SEQ ID NO: 180/SP0659 (28-end)SEQ ID NO: 181/SP0662 (29-276)SEQ ID NO: 182/SP0662 (300-end)SEQ ID NO: 183/SP0664 (103-629)SEQ ID NO: 184/SP0664 (630-1200)SEQ ID NO: 185/SP0664 (1201-end)SEQ ID NO: 186/SP0678 (23-end)SEQ ID NO: 187/SP0724 (35-end)SEQ ID NO: 188/SP0742 (43-end)SEQ ID NO: 189/SP0757 (44-451)SEQ ID NO: 190/SP0785 (33-end)SEQ ID NO: 191/SP0787 (43-290)SEQ ID NO: 192/SP0872 (30-end)SEQ ID NO: 193/SP0878 (245-end)SEQ ID NO: 194/SP0899 (31-end)SEQ ID NO: 195/SP1002 (22-end)SEQ ID NO: 196/SP1026 (24-end)SEQ ID NO: 197/SP1032 (22-end)SEQ ID NO: 198/SP1069 (34-end)SEQ ID NO: 199/SP1154 (155-694)SEQ ID NO: 200/SP1154 (695-1374)SEQ ID NO: 201/SP1154 (1375-end)SEQ ID NO: 202/SP1267 (25-end)SEQ ID NO: 203/SP1376 (32-end)SEQ ID NO: 204/SP1386 (33-end)SEQ ID NO: 205/SP1404 (31-end)SEQ ID NO: 206/SP1405 (19-end)SEQ ID NO: 207/SP1419 (27-end)SEQ ID NO: 208/SP1479 (40-end)SEQ ID NO: 209/SP1500 (27-end)SEQ ID NO: 210/SP1545 (29-end)SEQ ID NO: 211/SP1560 (28-end)SEQ ID NO: 212/SP1624 (1-217)SEQ ID NO: 213/SP1652 (62-397)SEQ ID NO: 214/SP1683 (65-end)SEQ ID NO: 215/SP1826 (36-end)SEQ ID NO: 216/SP1872 (40-end)SEQ ID NO: 217/SP1891 (40-end)SEQ ID NO: 218/SP1897 (30-end)SEQ ID NO: 219/SP1942 (37-end)SEQ ID NO: 220/SP1966 (25-end)SEQ ID NO: 221/SP1967 (30-end)SEQ ID NO: 222/SP1998 (51-end)SEQ ID NO: 223/SP2048 (40-end)SEQ ID NO: 224/SP2050 (35-end)SEQ ID NO: 225/SP2083 (192-end)SEQ ID NO: 226/SP2084 (30-end)SEQ ID NO: 227/SP2088 (30-end)SEQ ID NO: 70/SP2145 (1-end)SEQ ID NO: 228/SP2151 (25-end)SEQ ID NO: 229/SP2187 (32-end)SEQ ID NO: 230/SP2192 (224-end)SEQ ID NO: 231/SP2197 (30-end)SEQ ID NO: 232/SP2207 (30-end)SEQ ID NO: 234/SP2218 (106-end)

The invention claimed is:
 1. An immunogenic composition comprising afusion protein comprising (i) a biotin-binding protein or biotin-bindingportion thereof; and (ii) a therapeutically effective amount of anantigenic polypeptide comprising: the amino acid sequence of SEQ ID NO:34; or the amino acid sequence of SEQ ID NO:
 190. 2. The immunogeniccomposition of claim 1, wherein the biotin-binding protein isrhizavidin.
 3. The immunogenic composition of claim 1, furthercomprising a biotinylated polysaccharide.
 4. The immunogenic compositionof claim 3, wherein the polysaccharide is a pneumococcal polysaccharide.5. The immunogenic composition of claim 3, wherein the biotinylatedpolysaccharide is complexed with the fusion protein.
 6. The immunogeniccomposition of claim 3, further comprising an adjuvant.
 7. Theimmunogenic composition of claim 6, wherein the adjuvant is selectedfrom the group consisting of: cholera toxin, complete Freund's adjuvant(CFA), incomplete Freund's adjuvant (IFA) and alum.
 8. The immunogeniccomposition of claim 1, wherein the antigenic polypeptide consists ofthe amino acid sequence of SEQ ID NO: 34, or the amino acid sequence ofSEQ ID NO:
 190. 9. An immunogenic composition comprising a fusionprotein comprising (i) a biotin-binding protein or biotin-bindingportion thereof, and (ii) a therapeutically effective amount of anantigenic polypeptide comprising the amino acid sequence of SEQ ID NO:190.
 10. The immunogenic composition of claim 9, wherein thebiotin-binding protein is rhizavidin.
 11. The immunogenic composition ofclaim 9, further comprising a biotinylated polysaccharide.
 12. Theimmunogenic composition of claim 11, wherein the biotinylatedpolysaccharide is complexed with the fusion protein.
 13. The immunogeniccomposition of claim 9, wherein the polysaccharide is a pneumococcalpolysaccharide.
 14. The immunogenic composition of claim 9, furthercomprising an adjuvant.
 15. The immunogenic composition of claim 14,wherein the adjuvant is selected from the group consisting of: choleratoxin, complete Freund's adjuvant (CFA), incomplete Freund's adjuvant(IFA) and alum.
 16. The immunogenic composition of claim 9, wherein theantigenic polypeptide consists of the amino acid sequence of SEQ ID NO:190.